Compositions and methods for the therapy and diagnosis of pancreatic cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly pancreatic cancer, are disclosed. Illustrative compositions comprise one or more pancreatic tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly pancreatic cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Application No. 60/291,201 filed May 15, 2001, U.S. Provisional Application No. 60/265,447 filed Jan. 30, 2001, and U.S. Provisional Application No. 60/223,130 filed Aug. 7, 2000, incorporated in their entirety herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to therapy and diagnosis of cancer, such as pancreatic cancer. The invention is more specifically related to polypeptides, comprising at least a portion of a pancreatic tumor protein, and to polynucleotides encoding such polypeptides. Such polypeptides and polynucleotides are useful in pharmaceutical compositions, e.g., vaccines, and other compositions for the diagnosis and treatment of pancreatic cancer

[0004] 2. Description of the Related Art

[0005] Cancer is a significant health problem throughout the world. Although advances have been made in detection and therapy of cancer, no vaccine or other universally successful method for prevention and/or treatment is currently available. Current therapies, which are generally based on a combination of chemotherapy or surgery and radiation, continue to prove inadequate in many patients.

[0006] Pancreatic cancer is the fifth leading cause of cancer death in the United States. Current therapies for this common and difficult-to-treat disease include surgery and/or chemotherapy. Although 5-year survival rates after surgical removal of the pancreas and a large portion of the duodenum have improved, the procedure is only used on 9% of patients. Of these, the highest reported 5-year survival rate is in the range of 20%. Patients with advanced pancreatic cancer are treated primarily by chemotherapy. The objective of such therapy is to prolong patient survival. Surgery and irradiation are used as well to relieve pain and reduce organ blockage.

[0007] In spite of considerable research into therapies for these and other cancers, pancreatic cancer remains difficult to diagnose and treat effectively. Accordingly, there is a need in the art for improved methods for detecting and treating such cancers. The present invention fulfills these needs and further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides polynucleotide compositions comprising a sequence selected from the group consisting of:

[0009] (a) sequences provided in SEQ ID NO:1-149;

[0010] (b) complements of the sequences provided in SEQ ID NO:1-149;

[0011] (c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NO:1-149;

[0012] (d) sequences that hybridize to a sequence provided in SEQ ID NO:1-149 , under moderately stringent conditions;

[0013] (e) sequences having at least 75% identity to a sequence of SEQ ID NO:1-149;

[0014] (f) sequences having at least 90% identity to a sequence of SEQ ID NO:1-149; and

[0015] (g) degenerate variants of a sequence provided in SEQ ID NO:1-149.

[0016] In one preferred embodiment, the polynucleotide compositions of the invention are expressed in at least about 20%, more preferably in at least about 30%, and most preferably in at least about 50% of pancreatic tumors samples tested, at a level that is at least about 2-fold, preferably at least about 5-fold, and most preferably at least about 10-fold higher than that for normal tissues.

[0017] The present invention, in another aspect, provides polypeptide compositions comprising an amino acid sequence that is encoded by a polynucleotide sequence described above.

[0018] The present invention further provides polypeptide compositions comprising an amino acid sequence selected from the group consisting of sequences recited in SEQ ID NO:150-155.

[0019] In certain preferred embodiments, the polypeptides and/or polynucleotides of the present invention are immunogenic, i.e., they are capable of eliciting an immune response, particularly a humoral and/or cellular immune response, as further described herein.

[0020] The present invention further provides fragments, variants and/or derivatives of the disclosed polypeptide and/or polynucleotide sequences, wherein the fragments, variants and/or derivatives preferably have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% of the level of immunogenic activity of a polypeptide sequence set forth in SEQ ID NO:150-155 or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID NO:1-149.

[0021] The present invention further provides polynucleotides that encode a polypeptide described above, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.

[0022] Within other aspects, the present invention provides pharmaceutical compositions comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.

[0023] Within a related aspect of the present invention, the pharmaceutical compositions, e.g., vaccine compositions, are provided for prophylactic or therapeutic applications. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the invention and an immunostimulant, such as an adjuvant.

[0024] The present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of the present invention, or a fragment thereof; and (b) a physiologically acceptable carrier.

[0025] Within further aspects, the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient. Illustrative antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.

[0026] Within related aspects, pharmaceutical compositions are provided that comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) an immunostimulant.

[0027] The present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins, typically in the form of pharmaceutical compositions, e.g., vaccine compositions, comprising a physiologically acceptable carrier and/or an immunostimulant. The fusions proteins may comprise multiple immunogenic polypeptides or portions/variants thereof, as described herein, and may further comprise one or more polypeptide segments for facilitating the expression, purification and/or immunogenicity of the polypeptide(s).

[0028] Within further aspects, the present invention provides methods for stimulating an immune response in a patient, preferably a T cell response in a human patient, comprising administering a pharmaceutical composition described herein. The patient may be afflicted with pancreatic cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.

[0029] Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition as recited above. The patient may be afflicted with pancreatic cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.

[0030] The present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a polypeptide of the present invention, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the protein from the sample.

[0031] Within related aspects, methods are provided for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated as described above.

[0032] Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a polypeptide of the present invention, comprising contacting T cells with one or more of: (i) a polypeptide as described above; (ii) a polynucleotide encoding such a polypeptide; and/or (iii) an antigen presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Isolated T cell populations comprising T cells prepared as described above are also provided.

[0033] Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.

[0034] The present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of polypeptide disclosed herein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen-presenting cell that expressed such a polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient. Proliferated cells may, but need not, be cloned prior to administration to the patient.

[0035] Within further aspects, the present invention provides methods for determining the presence or absence of a cancer, preferably a pancreatic cancer, in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within preferred embodiments, the binding agent is an antibody, more preferably a monoclonal antibody.

[0036] The present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient. Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.

[0037] The present invention further provides, within other aspects, methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide of the present invention; (b) detecting in the sample a level of a polynucleotide, preferably mRNA, that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within certain embodiments, the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as recited above, or a complement of such a polynucleotide. Within other embodiments, the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.

[0038] In related aspects, methods are provided for monitoring the progression of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide of the present invention; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.

[0039] Within further aspects, the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.

[0040] These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THESEQUENCE IDENTIFIERS

[0041] SEQ ID NO:1 is the determined cDNA sequence for 58291contig

[0042] SEQ ID NO:2 is the determined cDNA sequence for 58292.1

[0043] SEQ ID NO:3 is the determined cDNA sequence for 58292.2

[0044] SEQ ID NO:4 is the determined cDNA sequence for 58295.1

[0045] SEQ ID NO:5 is the determined cDNA sequence for 58295.2

[0046] SEQ ID NO:6 is the determined cDNA sequence for 58296contig

[0047] SEQ ID NO:7 is the determined cDNA sequence for 58298.1

[0048] SEQ ID NO:8 is the determined cDNA sequence for 58298.2

[0049] SEQ ID NO:9 is the determined cDNA sequence for 58299contig

[0050] SEQ ID NO:10 is the determined cDNA sequence for 58303.1

[0051] SEQ ID NO:11 is the determined cDNA sequence for 58303.2

[0052] SEQ ID NO:12 is the determined cDNA sequence for 58304.1

[0053] SEQ ID NO:13 is the determined cDNA sequence for 58305contig

[0054] SEQ ID NO:14 is the determined cDNA sequence for 58308contig

[0055] SEQ ID NO:15 is the determined cDNA sequence for 58328.1

[0056] SEQ ID NO:16 is the determined cDNA sequence for 58330contig

[0057] SEQ ID NO:17 is the determined cDNA sequence for 58333.1

[0058] SEQ ID NO:18 is the determined cDNA sequence for 58333 .2

[0059] SEQ ID NO:19 is the determined cDNA sequence for 58334.2

[0060] SEQ ID NO:20 is the determined cDNA sequence for 58338.1

[0061] SEQ ID NO:21 is the determined cDNA sequence for TrypsinogenContig

[0062] SEQ ID NO:22 is the determined cDNA sequence for LipaseContig

[0063] SEQ ID NO:23 is the determined cDNA sequence for 59006.1

[0064] SEQ ID NO:24 is the determined cDNA sequence for 59019.1

[0065] SEQ ID NO:25 is the determined cDNA sequence for 59020.1

[0066] SEQ ID NO:26 is the determined cDNA sequence for 59023.1

[0067] SEQ ID NO:27 is the determined cDNA sequence for S9024.2

[0068] SEQ ID NO:28 is the determined cDNA sequence for 59026contig

[0069] SEQ ID NO:29 is the determined cDNA sequence for 59027.1

[0070] SEQ ID NO:30 is the determined cDNA sequence for 59027.2

[0071] SEQ ID NO:31 is the determined cDNA sequence for 59028contig

[0072] SEQ ID NO:32 is the determined cDNA sequence for 59032.1

[0073] SEQ ID NO:33 is the determined cDNA sequence for 59034.1

[0074] SEQ ID NO:34 is the determined cDNA sequence for 59035.1

[0075] SEQ ID NO:35 is the determined cDNA sequence for 59038.2

[0076] SEQ ID NO:36 is the determined cDNA sequence for 46.48contig

[0077] SEQ ID NO:37 is the determined cDNA sequence for 47.48contig

[0078] SEQ ID NO:38 is the determined cDNA sequence for 49.52contig

[0079] SEQ ID NO:39 is the determined cDNA sequence for 60226.1

[0080] SEQ ID NO:40 is the determined cDNA sequence for 60227.1

[0081] SEQ ID NO:41 is the determined cDNA sequence for 60228.1

[0082] SEQ ID NO:42 is the determined cDNA sequence for 60229.1

[0083] SEQ ID NO:43 is the determined cDNA sequence for 60234.1

[0084] SEQ ID NO:44 is the determined cDNA sequence for 60235.1

[0085] SEQ ID NO:45 is the determined cDNA sequence for 60236.1

[0086] SEQ ID NO:46 is the determined cDNA sequence for 60241.1

[0087] SEQ ID NO:47 is the determined cDNA sequence for 60993.1

[0088] SEQ ID NO:48 is the determined cDNA sequence for 60995.1

[0089] SEQ ID NO:49 is the determined cDNA sequence for 60997.1

[0090] SEQ ID NO:50 is the determined cDNA sequence for 60998.1

[0091] SEQ ID NO:51 is the determined cDNA sequence for 60999.1

[0092] SEQ ID NO:52 is the determined cDNA sequence for 61001.1

[0093] SEQ ID NO:53 is the determined cDNA sequence for 61004.1

[0094] SEQ ID NO:54 is the determined cDNA sequence for 61006.1

[0095] SEQ ID NO:55 is the determined cDNA sequence for 60246.1

[0096] SEQ ID NO:56 is the determined cDNA sequence for 60247.1

[0097] SEQ ID NO:57 is the determined cDNA sequence for 60248.1

[0098] SEQ ID NO:58 is the determined cDNA sequence for 60249.1

[0099] SEQ ID NO:59 is the determined cDNA sequence for 60250.1

[0100] SEQ ID NO:60 is the determined cDNA sequence for 60253.1

[0101] SEQ ID NO:61 is the determined cDNA sequence for 61008.1

[0102] SEQ ID NO:62 is the determined cDNA sequence for 61009.1

[0103] SEQ ID NO:63 is the determined cDNA sequence for 61011.1

[0104] SEQ ID NO:64 is the determined cDNA sequence for 61014.1

[0105] SEQ ID NO:65 is the determined cDNA sequence for 61016.1

[0106] SEQ ID NO:66 is the determined cDNA sequence for 61018.1

[0107] SEQ ID NO:67 is the determined cDNA sequence for 61020.1

[0108] SEQ ID NO:68 is the determined cDNA sequence for 61021.1

[0109] SEQ ID NO:69 is the determined cDNA sequence for 61022.1

[0110] SEQ ID NO:70 is the determined cDNA sequence for S2B CarboxypeptidaseContig.seq(1>207)

[0111] SEQ ID NO:71 is the determined cDNA sequence for S2B CollagenContig.seq(1>658)

[0112] SEQ ID NO:72 is the determined cDNA sequence for S2B VersicanContig.seq(1>550)

[0113] SEQ ID NO:73 is the determined cDNA sequence for 58316.1

[0114] SEQ ID NO:74 is the determined cDNA sequence for 58316.2

[0115] SEQ ID NO:75 is the determined cDNA sequence for 58317.1

[0116] SEQ ID NO:76 is the determined cDNA sequence for 58317.2

[0117] SEQ ID NO:77 is the determined cDNA sequence for 58318.1

[0118] SEQ ID NO:78 is the determined cDNA sequence for 58318.2

[0119] SEQ ID NO:79 is the determined cDNA sequence for 58319.1

[0120] SEQ ID NO:80 is the determined cDNA sequence for 58319.2

[0121] SEQ ID NO:81 is the determined cDNA sequence for 58321.1

[0122] SEQ ID NO:82 is the determined cDNA sequence for 58321.2

[0123] SEQ ID NO:83 is the determined cDNA sequence for 58322.2

[0124] SEQ ID NO:84 is the determined cDNA sequence for 58324.1

[0125] SEQ ID NO:85 is the determined cDNA sequence for 58324.2

[0126] SEQ ID NO:86 is the determined cDNA sequence for 58327.2

[0127] SEQ ID NO:87 is the determined cDNA sequence for 58329.1

[0128] SEQ ID NO:88 is the determined cDNA sequence for 58329.2

[0129] SEQ ID NO:89 is the determined cDNA sequence for 58330.1

[0130] SEQ ID NO:90 is the determined cDNA sequence for 58330.2

[0131] SEQ ID NO:91 is the determined cDNA sequence for 58331.1

[0132] SEQ ID NO:92 is the determined cDNA sequence for 58331.2

[0133] SEQ ID NO:93 is the determined cDNA sequence for 58335.1

[0134] SEQ ID NO:94 is the determined cDNA sequence for 58335.2

[0135] SEQ ID NO:95 is the determined cDNA sequence for 58336.1

[0136] SEQ ID NO:96 is the determined cDNA sequence for 58336.2

[0137] SEQ ID NO:97 is the determined cDNA sequence for 58771.1

[0138] SEQ ID NO:98 is the determined cDNA sequence for 58771.2

[0139] SEQ ID NO:99 is the determined cDNA sequence for 58772.1

[0140] SEQ ID NO:100 is the determined cDNA sequence for 58772.2

[0141] SEQ ID NO:101 is the determined cDNA sequence for 58773.1

[0142] SEQ ID NO:102 is the determined cDNA sequence for 58773.2

[0143] SEQ ID NO:103 is the determined cDNA sequence for 58774.1

[0144] SEQ ID NO:104 is the determined cDNA sequence for 58774.2

[0145] SEQ ID NO:105 is the determined cDNA sequence for 59034.1

[0146] SEQ ID NO:106 is the determined cDNA sequence for 59034.2

[0147] SEQ ID NO:107 is the determined cDNA sequence for 59038.1

[0148] SEQ ID NO:108 is the determined cDNA sequence for 59038.2

[0149] SEQ ID NO:109 is the determined cDNA sequence for 59046.1

[0150] SEQ ID NO:110 is the determined cDNA sequence for 59046.2

[0151] SEQ ID NO:111 is the determined cDNA sequence for 59047.1

[0152] SEQ ID NO:112 is the determined cDNA sequence for 59047.2

[0153] SEQ ID NO:113 is the determined cDNA sequence for 59048.1

[0154] SEQ ID NO:114 is the determined cDNA sequence for 59048.2

[0155] SEQ ID NO:115 is the determined cDNA sequence for 59049.1

[0156] SEQ ID NO:116 is the determined cDNA sequence for 59050.1

[0157] SEQ ID NO:117 is the determined cDNA sequence for 59050.2

[0158] SEQ ID NO:118 is the determined cDNA sequence for 59052.1

[0159] SEQ ID NO:119 is the determined cDNA sequence for 59052.2

[0160] SEQ ID NO:120 is the determined cDNA sequence for 60246.1

[0161] SEQ ID NO:121 is the determined cDNA sequence for 60250.1

[0162] SEQ ID NO:122 is the determined cDNA sequence for 61008.1

[0163] SEQ ID NO:123 is the determined cDNA sequence for 61016.1

[0164] SEQ ID NO:124 is the determined cDNA sequence for 61806.1

[0165] SEQ ID NO:125 is the determined cDNA sequence for 61806.2

[0166] SEQ ID NO:126 is the determined cDNA sequence for 61807.1

[0167] SEQ ID NO:127 is the determined cDNA sequence for 61807.2

[0168] SEQ ID NO:128 is the determined cDNA sequence for 61809.1

[0169] SEQ ID NO:129 is the determined cDNA sequence for 61809.2

[0170] SEQ ID NO:130 is the determined cDNA sequence for 61810.1

[0171] SEQ ID NO:131 is the determined cDNA sequence for 61810.2

[0172] SEQ ID NO:132 is the determined cDNA sequence for 61811.1

[0173] SEQ ID NO:133 is the determined cDNA sequence for 61811.2

[0174] SEQ ID NO:134 is the determined cDNA sequence for 62760.1

[0175] SEQ ID NO:135 is the determined cDNA sequence for 62760.2

[0176] SEQ ID NO:136 is the determined cDNA sequence for 62761.1

[0177] SEQ ID NO:137 is the determined cDNA sequence for 62761.2

[0178] SEQ ID NO:138 is the determined cDNA sequence for CarboxypeptidaseA2 Consensus.seq(1>197)

[0179] SEQ ID NO:139 is the determined cDNA sequence for LipaseConsensus. seq(1>938)

[0180] SEQ ID NO:140 is the determined cDNA sequence for ProteaseE Consensus.seq(1>256)

[0181] SEQ ID NO:141 is the determined cDNA sequence for PUMP 1 Consensus. seq (1>375)

[0182] SEQ ID NO:142 is the determined cDNA sequence for Trypsinogen Consensus.seq(1>567)

[0183] SEQ ID NO:143 is the determined cDNA sequence for Vimentin Consensus.seq(1>567)

[0184] SEQ ID NO:144 is the determined cDNA sequence for Carboxypeptidase A2.Genbank.seq(1>1306)

[0185] SEQ ID NO:145 is the determined cDNA sequence for Lipase.Genbank. seq(1>1471)

[0186] SEQ ID NO:146 is the determined cDNA sequence for ProteaseE. Genbank. seq(1>897)

[0187] SEQ ID NO:147 is the determined cDNA sequence for PUMP 1. Genbank . seq (1.1078)

[0188] SEQ ID NO:148 is the determined cDNA sequence for Trypsinogen. Genbank. seq(1>802)

[0189] SEQ ID NO:149 is the determined cDNA sequence for Vimentin .Genbank. seq (1>1749)

[0190] SEQ ID NO:150 is the amino acid sequence of CarboxypeptidaseA2 .Protein.pro

[0191] SEQ ID NO:151 is the amino acid sequence of Lipase.Protein.pro

[0192] SEQ ID NO:152 is the amino acid sequence of ProteaseE.Protein.pro

[0193] SEQ ID NO:153 is the amino acid sequence of PUMP1 .Protein.pro

[0194] SEQ ID NO:154 is the amino acid sequence of Trypsinogen.Protein.pro

[0195] SEQ ID NO:155 is the amino acid sequence of Vimentin.Protein.pro

DETAILED DESCRIPTION OF THE INVENTION

[0196] The present invention is directed generally to compositions and their use in the therapy and diagnosis of cancer, particularly pancreatic cancer. As described further below, illustrative compositions of the present invention include, but are not restricted to, polypeptides, particularly immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (e.g. T cells).

[0197] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

[0198] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0199] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

[0200] Polypeptide Compositions

[0201] As used herein, the term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.

[0202] Particularly illustrative polypeptides of the present invention comprise those encoded by a polynucleotide sequence set forth in any one of SEQ ID NO: 1-149, or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions, to a polynucleotide sequence set forth in any one of SEQ ID NO: 1-149 Certain other illustrative polypeptides of the invention comprise amino acid sequences as set forth in any one of SEQ ID NO: 150-155.

[0203] The polypeptides of the present invention are sometimes herein referred to as pancreatic tumor proteins or pancreatic tumor polypeptides, as an indication that their identification has been based at least in part upon their increased levels of expression in pancreatic tumor samples. Thus, a “pancreatic tumor polypeptide” or “pancreatic tumor protein,” refers generally to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide, that is expressed in a substantial proportion of pancreatic tumor samples, for example preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50% or more of pancreatic tumor samples tested, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in normal tissues, as determined using a representative assay provided herein. A pancreatic tumor polypeptide sequence of the invention, based upon its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.

[0204] In certain preferred embodiments, the polypeptides of the invention are immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with pancreatic cancer. Screening for immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, ¹²⁵I-labeled Protein A.

[0205] As would be recognized by the skilled artisan, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention. An “immunogenic portion,” as used herein, is a fragment of an immunogenic polypeptide of the invention that itself is immunologically reactive (i.e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide. Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are “antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well-known techniques.

[0206] In one preferred embodiment, an immunogenic portion of a polypeptide of the present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Preferably, the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70% and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide. In some instances, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.

[0207] In certain other embodiments, illustrative immunogenic portions may include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted. Other illustrative immunogenic portions will contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.

[0208] In another embodiment, a polypeptide composition of the invention may also comprise one or more polypeptides that are immunologically reactive with T cells and/or antibodies generated against a polypeptide of the invention, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof.

[0209] In another embodiment of the invention, polypeptides are provided that comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.

[0210] The present invention, in another aspect, provides polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide compositions set forth herein, such as those set forth in SEQ ID NO: 150-155, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID NO: 1-149

[0211] In another aspect, the present invention provides variants of the polypeptide compositions described herein. Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein.

[0212] In one preferred embodiment, the polypeptide fragments and variants provide by the present invention are immunologically reactive with an antibody and/or T-cell that reacts with a full-length polypeptide specifically set for the herein.

[0213] In another preferred embodiment, the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90% or more of that exhibited by a full-length polypeptide sequence specifically set forth herein.

[0214] A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity as described herein and/or using any of a number of techniques well known in the art.

[0215] For example, certain illustrative variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other illustrative variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.

[0216] In many instances, a variant will contain conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, immunogenic variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table 1.

[0217] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UGA UCC UGG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0218] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0219] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0220] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0221] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0222] In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0223] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

[0224] As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0225] When comparing polypeptide sequences, two sequences are said to be “identical” if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0226] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0227] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0228] One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

[0229] In one preferred approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0230] Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.

[0231] Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

[0232] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0233] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide.

[0234] The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J Med., 336:86-91, 1997).

[0235] In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. patent application Ser. No. 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. patent application Ser. No. 60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as a soluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ra12 polypeptide or a portion thereof.

[0236] Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

[0237] In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

[0238] Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the invention, when fused with this targeting signal, will associate more efficiently with MHC class II molecules and thereby provide enhanced in vivo stimulation of CD4⁺ T-cells specific for the polypeptide.

[0239] Polypeptides of the invention are prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further described below. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

[0240] In general, polypeptide compositions (including fusion polypeptides) of the invention are isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.

[0241] Polynucleotide Compositions

[0242] The present invention, in other aspects, provides polynucleotide compositions. The terms “DNA” and “polynucleotide” are used essentially interchangeably herein to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. “Isolated,” as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

[0243] As will be understood by those skilled in the art, the polynucleotide compositions of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.

[0244] As will be also recognized by the skilled artisan, polynucleotides of the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

[0245] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide/protein of the invention or a portion thereof) or may comprise a sequence that encodes a variant or derivative, preferably and immunogenic variant or derivative, of such a sequence.

[0246] Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that comprise some or all of a polynucleotide sequence set forth in any one of SEQ ID NO: 1-149 , complements of a polynucleotide sequence set forth in any one of SEQ ID NO: 1-149 , and degenerate variants of a polynucleotide sequence set forth in any one of SEQ ID NO: 1-149 . In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above.

[0247] In other related embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID NO: -1-149 for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0248] Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein). The term “variants” should also be understood to encompasses homologous genes of xenogenic origin.

[0249] In additional embodiments, the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that “intermediate lengths”, in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.

[0250] In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5× SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5× SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2× SSC containing 0.1% SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65° C. or 65-70° C.

[0251] In certain preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.

[0252] The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.

[0253] When comparing polynucleotide sequences, two sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0254] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M .O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0255] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0256] One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

[0257] Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0258] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

[0259] Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of immunogenic variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0260] Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0261] In certain embodiments of the present invention, the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

[0262] As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

[0263] In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

[0264] The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

[0265] As used herein, the term “oligonucleotide directed mutagenesis procedure” refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term “oligonucleotide directed mutagenesis procedure” is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224, specifically incorporated herein by reference in its entirety.

[0266] In another approach for the production of polypeptide variants of the present invention, recursive sequence recombination, as described in U.S. Pat. No. 5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to “evolve” individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.

[0267] In other embodiments of the present invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.

[0268] The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

[0269] Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.

[0270] The use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.

[0271] Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.

[0272] Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Pat. No. 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

[0273] The nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50° C. to about 70° C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.

[0274] Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

[0275] According to another embodiment of the present invention, polynucleotide compositions comprising antisense oligonucleotides are provided. Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al., Science. 1988 June 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 June 15;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683).

[0276] Therefore, in certain embodiments, the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein. Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense compositions may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA, are those which are at or near the AUG translation initiation codon, and those sequences which are substantially complementary to 5′ regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software and/or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0277] The use of an antisense delivery method employing a short peptide vector, termed MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 July 15;25(14):2730-6). It has been demonstrated that several molecules of the MPG peptide coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.

[0278] According to another embodiment of the invention, the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of the tumor polypeptides and proteins of the present invention in tumor cells. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 April 24;49(2):211-20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. 1981 December;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 December 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374):173-6). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.

[0279] Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0280] The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., Proc Natl Acad Sci U S A. 1992 August 15;89(16):7305-9). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.

[0281] The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis 6 virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al Nucleic Acids Res. 1992 September 11;20(17):4559-65. Examples of hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz, Biochemistry 1989 June 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 January 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis 6 virus motif is described by Perrotta and Been, Biochemistry. 1992 December 1;31(47):11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al., Cell. 1983 December;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci U S A. 1991 October 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 March 23;32(11):2795-9); and an example of the Group I intron is described in (U.S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.

[0282] Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.

[0283] Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

[0284] Sullivan et al (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.

[0285] Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells Ribozymes expressed from such promoters have been shown to function in mammalian cells. Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).

[0286] In another embodiment of the invention, peptide nucleic acids (PNAs) compositions are provided. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 June;15(6):224-9). As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell to which such PNA compositions have been administered.

[0287] PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., Science 1991 December 6;254(5037):1497-500; Hanvey et al., Science. 1992 November 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.

[0288] PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., Bioorg Med Chem. 1995 April;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

[0289] As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.

[0290] Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem. 1995 April;3(4):437-45; Petersen et al., J Pept Sci. 1995 May-June;1(3):175-83; Orum et al., Biotechniques. 1995 September;19(3):472-80; Footer et al., Biochemistry. 1996 August 20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 August 11;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci U S A. 1995 June 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci U S A. 1995 March 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 August 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U S A. 1997 November 11;94(23):12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

[0291] Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. 1993 December 15;65(24):3545-9) and Jensen et al. (Biochemistry. 1997 April 22;36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcore™ technology.

[0292] Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.

[0293] Polynucleotide Identification, Characterization and Expression

[0294] Polynucleotides compositions of the present invention may be identified, prepared and/or manipulated using any of a variety of well established techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, and other like references). For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using the microarray technology of Affymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.

[0295] Many template dependent processes are available to amplify a target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

[0296] Any of a number of other template dependent processes, many of which are variations of the PCR™ amplification technique, are readily known and available in the art. Illustratively, some such methods include the ligase chain reaction (referred to as LCR), described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in Great Britain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822 describes a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Other amplification methods such as “RACE” (Frohman, 1990), and “one-sided PCR” (Ohara, 1989) are also well-known to those of skill in the art.

[0297] An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a tumor cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5′ and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5′ sequences.

[0298] For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with ³²P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.

[0299] Alternatively, amplification techniques, such as those described above, can be useful for obtaining a full length coding sequence from a partial cDNA sequence. One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as “rapid amplification of cDNA ends” or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5′ and 3′ of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

[0300] In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.

[0301] In other embodiments of the invention, polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

[0302] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0303] Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.

[0304] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.

[0305] Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0306] A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

[0307] In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

[0308] A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0309] The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

[0310] In bacterial systems, any of a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0311] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0312] In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0313] An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. fiugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

[0314] In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0315] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

[0316] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

[0317] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

[0318] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol Biol. 55:121-131).

[0319] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0320] Alternatively, host cells that contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

[0321] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

[0322] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0323] Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0324] In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

[0325] Antibody Compositions Fragments Thereof and Other Binding Agents

[0326] According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that exhibit immunological binding to a tumor polypeptide disclosed herein, or to a portion, variant or derivative thereof. An antibody, or antigen-binding fragment thereof, is said to “specifically bind,” “immunogically bind,” and/or is “immunologically reactive” to a polypeptide of the invention if it reacts at a detectable level (within, for example, an ELISA assay) with the polypeptide, and does not react detectably with unrelated polypeptides under similar conditions.

[0327] Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (K_(on)) and the “off rate constant” (K_(off)) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K_(off)/K_(on) enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0328] An “antigen-binding site,” or “binding portion” of an antibody refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”

[0329] Binding agents may be further capable of differentiating between patients with and without a cancer, such as pancreatic cancer, using the representative assays provided herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. Preferably, a statistically significant number of samples with and without the disease will be assayed. Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.

[0330] Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

[0331] Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

[0332] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

[0333] A number of therapeutically useful molecules are known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the “F(ab)” fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the “F(ab′)₂ ” fragment which comprises both antigen-binding sites. An “Fv” fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V_(H)::V_(L)heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0334] A single chain Fv (“sFv”) polypeptide is a covalently linked V_(H)::V_(L) heterodimer which is expressed from a gene fusion including V_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

[0335] Each of the above-described molecules includes a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain FR set which provide support to the CDRS and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

[0336] As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRS. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures—regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[0337] A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No. 519,596, published Dec. 23, 1992). These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.

[0338] As used herein, the terms “veneered FRs” and “recombinantly veneered FRs” refer to the selective replacement of FR residues from, e.g., a rodent heavy or light chain V region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen-binding site which retains substantially all of the native FR polypeptide folding structure. Veneering techniques are based on the understanding that the ligand binding characteristics of an antigen-binding site are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other, and their interaction with the rest of the V region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.

[0339] The process of veneering makes use of the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1987), updates to the Kabat database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities of V region amino acids can be deduced from the known three-dimensional structure for human and murine antibody fragments. There are two general steps in veneering a murine antigen-binding site. Initially, the FRs of the variable domains of an antibody molecule of interest are compared with corresponding FR sequences of human variable domains obtained from the above-identified sources. The most homologous human V regions are then compared residue by residue to corresponding murine amino acids. The residues in the murine FR which differ from the human counterpart are replaced by the residues present in the human moiety using recombinant techniques well known in the art. Residue switching is only carried out with moieties which are at least partially exposed (solvent accessible), and care is exercised in the replacement of amino acid residues which may have a significant effect on the tertiary structure of V region domains, such as proline, glycine and charged amino acids.

[0340] In this manner, the resultant “veneered” murine antigen-binding sites are thus designed to retain the murine CDR residues, the residues substantially adjacent to the CDRs, the residues identified as buried or mostly buried (solvent inaccessible), the residues believed to participate in non-covalent (e.g., electrostatic and hydrophobic) contacts between heavy and light chain domains, and the residues from conserved structural regions of the FRs which are believed to influence the “canonical” tertiary structures of the CDR loops. These design criteria are then used to prepare recombinant nucleotide sequences which combine the CDRs of both the heavy and light chain of a murine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies which exhibit the antigen specificity of the murine antibody molecule.

[0341] In another embodiment of the invention, monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.

[0342] A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0343] Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.

[0344] It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.

[0345] Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).

[0346] It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.

[0347] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.

[0348] T Cell Compositions

[0349] The present invention, in another aspect, provides T cells specific for a tumor polypeptide disclosed herein, or for a variant or derivative thereof. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the Isolex™ System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

[0350] T cells may be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide of interest. Preferably, a tumor polypeptide or polynucleotide of the invention is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.

[0351] T cells are considered to be specific for a polypeptide of the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a tumor polypeptide (100 μg/ml −100 μg/ml, preferably 200 μg/ml −25 μg/ml) for 3-7 days will typically result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to a tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumor polypeptide-specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.

[0352] For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate in response to a tumor polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a tumor polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a tumor polypeptide. Alternatively, one or more T cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.

[0353] Pharmaceutical Compositions

[0354] In additional embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody compositions disclosed herein in pharmaceutically-acceptable carriers for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.

[0355] It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may farther comprise substituted or derivatized RNA or DNA compositions.

[0356] Therefore, in another aspect of the present invention, pharmaceutical compositions are provided comprising one or more of the polynucleotide, polypeptide, antibody, and/or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise immunogenic polynucleotide and/or polypeptide compositions of the invention for use in prophylactic and theraputic vaccine applications. Vaccine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995). Generally, such compositions will comprise one or more polynucleotide and/or polypeptide compositions of the present invention in combination with one or more immunostimulants.

[0357] It will be apparent that any of the pharmaceutical compositions described herein can contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts can be prepared, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

[0358] In another embodiment, illustrative immunogenic compositions, e.g., vaccine compositions, of the present invention comprise DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered within any of a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate polynucleotide expression systems will, of course, contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and terminating signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.

[0359] Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems. In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0360] In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).

[0361] Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0362] Additional viral vectors useful for delivering the polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup.(−) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0363] A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0364] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0365] Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Pat. Nos. 5,505,947 and 5,643,576.

[0366] Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.

[0367] Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.

[0368] In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.

[0369] In another embodiment of the invention, a polynucleotide is administered/delivered as “naked” DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

[0370] In still another embodiment, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, Wis.), some examples of which are described in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.

[0371] In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, Oreg.), some examples of which are described in U.S. Pat. Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

[0372] According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2,-7,-12, and other like growth factors, may also be used as adjuvants.

[0373] Within certain embodiments of the invention, the adjuvant composition is preferably one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNF-α, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1 -type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

[0374] Certain preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, P-escin, or digitonin.

[0375] Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such as Carbopol^(R) to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.

[0376] In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

[0377] Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.

[0378] Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.

[0379] Other preferred adjuvants include adjuvant molecules of the general formula

(I): HO(CH₂CH₂O)_(n)—A—R,

[0380] wherein, n is 1-50, A is a bond or —(O)—, R is C₁₋₅₀ alkyl or Phenyl C₁₋₅₀ alkyl.

[0381] One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂ alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12^(th) edition: entry 7717). These adjuvant molecules are described in WO 99/52549.

[0382] The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.

[0383] According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0384] Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).

[0385] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

[0386] Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0387] APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

[0388] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will typically vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.

[0389] Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0390] In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems, such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.

[0391] The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate.

[0392] The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0393] The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.

[0394] In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0395] The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature 1997 March 27;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0396] Typically, these formulations will contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0397] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

[0398] In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.

[0399] Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0400] In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.

[0401] In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

[0402] The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

[0403] In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 March 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045.

[0404] In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.

[0405] The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 July;16(7):307-21; Takakura, Nippon Rinsho 1998 March;56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 August;35(8):801-9; Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety).

[0406] Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, J Biol Chem. 1990 September 25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 April;9(3):221-9). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.

[0407] In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

[0408] Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998 December;24(12):1113-28). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 January 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

[0409] Cancer Therapeutic Methods

[0410] In further aspects of the present invention, the pharmaceutical compositions described herein may be used for the treatment of cancer, particularly for the immunotherapy of pancreatic cancer. Within such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. As discussed above, administration of the pharmaceutical compositions may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.

[0411] Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).

[0412] Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Pat. No. 4,918,164) for passive immunotherapy.

[0413] Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).

[0414] Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.

[0415] Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 μg to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

[0416] In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

[0417] Cancer Detection and Diagnostic Compositions Methods and Kits

[0418] In general, a cancer may be detected in a patient based on the presence of one or more pancreatic tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as pancreatic cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample.

[0419] Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer. In general, a pancreatic tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.

[0420] In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length pancreatic tumor proteins and polypeptide portions thereof to which the binding agent binds, as described above.

[0421] The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent.

[0422] Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

[0423] In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

[0424] More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with pancreatic cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

[0425] Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited above.

[0426] The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

[0427] To determine the presence or absence of a cancer, such as pancreatic cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.

[0428] In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

[0429] Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such tumor protein specific antibodies may correlate with the presence of a cancer.

[0430] A cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample. Within certain methods, a biological sample comprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37° C. with polypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of pancreatic tumor polypeptide to serve as a control. For CD4⁺ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8⁺ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer in the patient.

[0431] As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a pancreatic tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (P CR) based assay to amplify a portion of a tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.

[0432] To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a tumor protein of the invention that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence as disclosed herein. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

[0433] One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.

[0434] In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.

[0435] Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.

[0436] As noted above, to improve sensitivity, multiple tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.

[0437] The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.

[0438] Alternatively, a kit may be designed to detect the level of mRNA encoding a tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a tumor protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a tumor protein.

[0439] The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1 Identification of Pancreatic Tumor Protein cDNAs

[0440] This Example illustrates the identification of cDNA molecules encoding pancreatic tumor proteins.

[0441] Pancreas tumor grade II-III and III-IV subtractions 1 and 2 were generated by conventional biotin-streptavidin subtraction. The testers were cut with BamH I and Xho I while all drivers were cut with EcoR I, Not I, and Sfu I. For PanT3-4.S2, the spike was digested with Nco I, Spe I, and Apa I. One overnight hybridization of tester and driver (/spike) was performed at 68° C. and followed by the first biotin-streptavidin subtraction. Another 2-hour hybridization at 68° C. was followed by a second subtraction. cDNA remaining after the two subtractions was ligated into a BamH I/Xho I-cut pBC-SK+vector.

[0442] The following Table 2 summarizes the reaction conditions employed in performing each of the pancreas tumor library subtractions of the present invention: TABLE 2 PANCREAS TUMOR SUBTRACTION CONDITIONS Pancreas Tumor Pancreas Tumor Pancreas Tumor Pancreas Tumor Grade II-III Grade III-IV Grade II-III Grade III-IV Subtraction 1 Subtraction 1 Subtraction 2 and Subtraction 2 and (PanT2-3.S1) in (PanT3-4.S1) in 2B (PanT2-3.S2 2B (PanT3-4.S2 pBC-SK + (754- pBC-SK + (754- & 2B) in pBC- & 2B) in pBC- 72) 72) SK + (754-88) SK + (754-88) Tester: 10 μg Pancreas 10 μg Pancreas 8 μg Pancreas 10 μg Pancreas Tumor Grade II- Tumor Grade III- Tumor Grade II- Tumor Grade III- III Library in IV Library in III Library in IV Library in pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) Driver: 25 μg Normal 40 μg Liver and 25 μg Normal 32 μg Liver and Pancreas in Salivary Gland in Pancreas in Salivary Gland in pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) 25 μg Liver and 60 μg Pooled 25 μg Liver and 48 μg Pooled Salivary Gland in Driver in Salivary Gland in Driver in pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) pcDNA3.1 (+) (liver, pancreas, liver, pancreas, 50 μg Pooled skin, bone 50 μg Pooled skin, bone Driver in marrow, resting Driver in marrow, resting pcDNA3.1 (+) PBMC, stomach, pcDNA3.1 (+) PBMC, stomach, (liver, pancreas, whole brain) (liver, pancreas, whole brain) skin, bone skin, bone marrow, resting marrow, resting PBMC, stomach, PBMC, stomach, whole brain) whole brain) Spike: 10 μg Human pancreatic trypsinogen (TRY2) in pBC- SK+ 10 μg Human triglyceride lipase in pBC-SK+

[0443] cDNA sequences isolated from these subtracted libraries represent cDNAs that are over-expressed in pancreas tumors and were searched against public databases including Genbank. Those sequences showing some degree of similarity to known sequences in Genbank are shown in Tables 3 and 4. Several cDNAs that showed no significant similiarity to any known sequences were identified and are listed in Table 5. Multiple sequences from these subtractions align to form consensus (contig) sequences shown in Table 6. The Genbank sequences from Table 6 are disclosed in SEQ ID NO: 144-149 (cDNA) and 150-155 (amino acid). TABLE 3 cDNAs ISOLATED FROM PANCREAS TUMOR GRADE II-III SUBTRACTIONS 1 THROUGH 2B (PANT2-3.S1 THROUGH PANT2-3.S2B) SEQ ID NO: Clone Identifier Library/Exp Genbank Nucleotide Database Search Results 1 58291contig PanT2-3.S1 Homo sapiens ribosomal protein L13a (RPL13A), mRNA 2 & 3 58292 PanT2-3.S1 Homo sapiens cDNA FLJ10423 fis, clone NT2RP1000259 4 & 5 58295 PanT2-3.S1 Human mRNA for pro-alpha-1 type 3 collagen 6 58296contig PanT2-3.S1 Homo sapiens mRNA for KIAA0310 protein, partial cds 7 & 8 58298 PanT2-3.S1 Homo sapiens ribosomal protein, large, P0 (RPLP0) mRNA 10 & 11 58303 PanT2-3.S1 Human cathepsin D mRNA, complete cds 12 58304 PanT2-3.S1 Human novel gene mRNA, complete cds 13 58305contig PanT2-3.S1 Homo sapiens chondroitin sulfate proteoglycan 2 (versican) (CSPG2) mRNA 14 58308contig PanT2-3.S1 Human mRNA for collagenase (E.C. 3.4.24) 23 59006 PanT2-3.S2 Human heterogeneous ribonucleoprotein A0 mRNA, complete cds 24 59019 PanT2-3.S2 Homo sapiens collagen, type I, alpha 1 (COL1A1) mRNA 25 59020 PanT2-3.S2 Homo sapiens dUTPase (DUT) gene, exons 1 and 2 26 59023 PanT2-3.S2 H. sapiens 28S rRNA V8 region (MRC5V2-2) 27 59024 PanT2-3.S2 Human versican V2 core protein precursor splice-variant mRNA, complete cds 28 59026contig PanT2-3.S2 Human mRNA for pro-alpha-1 type 3 collagen 31 59028contig PanT2-3.S2 Homo sapiens calcitonin receptor-like receptor activity modifying protein 1 (RAMP1), mRNA 39 60226 PanT2-3.S2B Homo sapiens cathepsin B (CTSB) mRNA 41 60228 PanT2-3.S2B Homo sapiens actin, beta (ACTB), mRNA 42 60229 PanT2-3.S2B Homo sapiens cDNA FLJ11718 fis, clone HEMBA1005252, highly similar to Homo sapiens mRNA for KIAA0585 protein 43 60234 PanT2-3.S2B Homo sapiens epoxide hydrolase 1, microsomal (xenobiotic) (EPHX1), mRNA 46 60241 PanT2-3.S2B Homo sapiens reticulocalbin 1, EF-hand calcium binding domain (RCN1), mRNA 47 60993 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 48 60995 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 49 60997 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 50 60998 PanT2-3.S2B Homo sapiens chondroitin sulfate proteoglycan 2 (versican) (CSPG2) mRNA 51 60999 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 52 61001 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 53 61004 PanT2-3.S2B Human mRNA for pro-alpha-1 type 3 collagen 54 61006 PanT2-3.S2B Human erg protein (ets-related gene) mRNA, complete cds

[0444] TABLE 4 cDNAS ISOLATED FROM PANCREAS TUMOR GRADE III-IV SUBTRACTIONS 1-2B (PANT3-4.S1 THROUGH PANT3-4.S2B) Clone SEQ ID NO: Identifier Library/Exp Genbank Nucleotide Database Search Results 15 58328 PanT3-4S1 H. sapiens 28S rRNA V8 region 16 58330contig PanT3-4S1 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 17 58333 PanT3-4S1 Human mitochondrial genome 18 58333 PanT3-4S1 Human 28S ribosomal RNA gene 19 58334 PanT3-4S1 Homo sapiens guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 (GNB2L1), mRNA 20 58338 PanT3-4S1 Homo sapiens upregulated by 1,25-dihydorxyvitamin D-3 (VDUP1), mRNA 21 Trypsinogen PanT3-4S1 Human pancreatic trypsinogen (TRY2) mRNA, complete Contig cds 22 LipaseContig PanT3-4S1 Human triglyceride lipase mRNA, complete cds 32 59032 PanT3-4S2 caldecrin = serum calcium-decreasing factor [human, pancreas, mRNA Partial, 894 nt] 33, 105, 106 59034 PanT3-4.S2 Homo sapiens pancreatic lipase (PNLIP), mRNA 34 59035 PanT3-4S2 Homo sapiens pancreatitis-associated protein (PAP), mRNA 35, 107, 108 59038 PanT3-4.S2 Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 36 46.48contig PanT3-4S2 Homo sapiens vimentin (VIM) mRNA 37 47.50contig PanT3-4S2 Human pump-1 mRNA homolog. to metalloproteinase, collagenase andstromelysin 38 49.52contig PanT3-4S2 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 55 & 120 60246 PanT3-4S2B Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 56 60247 PanT3-4.S2B Human mRNA for pro-alpha-1 type 3 collagen 58 60249 PanT3-4.S2B Homo sapiens regenerating islet-derived 1 beta (pancreatic stoneoprotein, pancreatic thread protein) (REG1B), mRNA 59 & 121 60250 PanT3-4S2B Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 60 60253 PanT3-4.S2B Homo sapiens BAC clone CTB-114B19 from 7q31.1, complete sequence 61 & 122 61008 PanT3-4S2B Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 62 61009 PanT3-4.S2B Human mRNA for pro-alpha-1 type 3 collagen 63 61011 PanT3-4.S2B Homo sapiens cytochrome b gene, complete cds; mitochondrial gene for mitochondrial product 64 61014 PanT3-4.S2B Homo sapiens chondroitin sulfate proteoglycan 2 (versican) (CSPG2), mRNA 66 61018 PanT3-4.S2B Human mRNA for pro-alpha-1 type 3 collagen 67 61020 PanT3-4.S2B H. sapiens 28S rRNA V8 region (MRC5-9) 68 61021 PanT3-4.S2B Human mRNA for pro-alpha-1 type 3 collagen 69 61022 PanT3-4.S2B Human mRNA for pro-alpha-1 type 3 collagen 73 & 74 58316 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 75 & 76 58317 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 77 & 78 58318 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 79 & 80 58319 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 81 & 82 58321 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 83 58322 PanT3-4.S1 Human pancreatic trypsinogen (TRY2) mRNA, complete cds 84 & 85 58324 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 86 58327 PanT3-4.S1 Human pancreatic trypsinogen (TRY2) mRNA, complete cds 87 & 88 58329 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 89 & 90 58330 PanT3-4.S1 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 91 & 92 58331 PanT3-4.S1 Human pancreatic trypsinogen (TRY2) mRNA, complete cds 93 & 94 58335 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 65 & 123 61016 PanT3-4S2B Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 95 & 96 58336 PanT3-4.S1 Homo sapiens pancreatic lipase (PNLIP), mRNA 97 & 98 58771 754-86 Human pancreatic trypsinogen (TRY2) mRNA, complete cds 99 & 100 58772 754-86 Human pancreatic trypsinogen (TRY2) mRNA, complete cds 101 & 102 58773 754-86 Homo sapiens pancreatic lipase (PNLIP), mRNA 103 & 104 58774 754-86 Homo sapiens pancreatic lipase (PNLIP), mRNA 109 & 110 59046 PanT3-4.S2 Human vimentin gene, complete cds 111 & 112 59047 PanT3-4.S2 Human pump-1 mRNA homolog. to metalloproteinase, collagenase and stromelysin 113 & 114 59048 PanT3-4.S2 Human vimentin gene, complete cds 115 59049 PanT3-4.S2 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 116 & 117 59050 PanT3-4.S2 Human pump-1 mRNA homolog. to metalloproteinase, collagenase and stromelysm 118 & 119 59052 PanT3-4.S2 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 124 & 125 61806 754-100 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 126 & 127 61807 754-100 Homo sapiens mRNA for pancreatic protease E precursor, complete cds 128 & 129 61809 754-100 Human pump-1 mRNA homolog. to metalloproteinase, collagenase and stromelysin 130 & 131 61810 754-100 Human vimentin gene, complete cds 132 & 133 61811 754-100 Human vimentin gene, complete cds 134 & 135 62760 754-130 Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA 136 & 137 62761 754-130 Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA

[0445] TABLE 5 cDNAs IDENTIFIED IN PANT2-3 AND PANT3-4 SUBTRACTED LIBRARIES THAT SHOW NO SIGNIFICANT SIMILARITY TO KNOWN SEQUENCES SEQ ID NO: Clone Identifier Library/Exp Genbank Nucleotide Database Search Results  9 58299contig PanT2-3.S1 29 & 30 59027 PanT2-3.S2 40 60227 PanT2-3.S2B 44 60235 PanT2-3.S2B 92% identity with Human hexokinase II pseudogene, complete cds 45 60236 PanT2-3.S2B 94% identity to Homo sapiens clone 77u-c10 immunoglobulin heavy chain variable region precursor (IgH) mRNA, partial cds 57 60248 PanT3-4.S2B

[0446] TABLE 6 MULTIPLE SEQUENCES FROM PANT2-3.S2B AND PANT3-4.S1 THROUGH PANT3-4.S2B ALIGN TO FORM 9 CONTIGS SEQ ID NO: (amino acid SEQ ID NO:) Clone Identifier Genbank Nucleotide Database Search Results 70 S2BCarboxypeptidas Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2) mRNA eContig 71 S2BCollagen Human mRNA for pro-alpha-1 type 3 collagen Contig 72 S2B VersicanContig Homo sapiens chondroitin sulfate proteoglycan 2 (versican) (CSPG2), mRNA 138 CarboxypeptidaseA2 Homo sapiens carboxypeptidase A2 (pancreatic) (CPA2), mRNA Consensus 139 LipaseConsensus Homo sapiens pancreatic lipase (PNLIP), mRNA 140 ProteaseE Homo sapiens mRNA for pancreatic protease E precursor, complete Consensus cds 141 PUMP1 Consensus Human pump-1 mRNA homolog. to metalloproteinase, collagenase and stromelysin 142 Trypsinogen Human pancreatic trypsinogen (TRY2) mRNA, complete cds Consensus 143 Vimentin Consensus Human vimentin gene, complete cds

Example 2 Analysis of Expression of Pancreatic Tumor cDNAs Using Microarray Technology

[0447] In additional studies, clones isolated from the subtraction libraries described in Example 1 were further evaluated for over-expression in specific tumor tissues by microarray analysis. Using this approach, cDNA sequences were PCR amplified and their mRNA expression profiles in tumor and normal tissues were examined using cDNA microarray technology essentially as described (Shena, M. et al., 1995 Science 270:467 70). In brief, the clones were arrayed onto glass slides as multiple replicas, with each location corresponding to a unique cDNA clone (as many as 5500 clones can be arrayed on a single slide, or chip). Each chip was hybridized with a pair of cDNA probes that were fluorescence-labeled with Cy3 and Cy5, respectively. Typically, 1 μg of polyA⁺ RNA was used to generate each cDNA probe. After hybridization, the chips were scanned and the fluorescence intensity recorded for both Cy3 and Cy5 channels. There were multiple built-in quality control steps. First, the probe quality was monitored using a panel of ubiquitously expressed genes. Secondly, the control plate also included yeast DNA fragments of which complementary RNA may be spiked into the probe synthesis for measuring the quality of the probe and the sensitivity of the analysis. Currently, the technology offers a sensitivity of 1 in 100,000 copies of mRNA. Finally, the reproducibility of this technology was ensured by including duplicated control cDNA elements at different locations.

[0448] Two clones were confirmed by microarray to be over-expressed by more than 4-fold in pancreas tumor tissue as compared to a panel of normal tissues. Pn628S (SEQ ID NO:101 and 102; also referred to as clone identifier 58773, Table 4) had an expression ratio of 5.56 (mean pancreas tumors/mean normals without pancreas) and <2 (mean pancreas tumors/mean normals including pancreas). This clone showed some degree of homology to Homo sapiens pancreatic lipase (PNLIP) mRNA in searches against the GenBank database (see Table 4). Pn630S (SEQ ID NO:128 and 129; also referred to as clone identifier 61809, Table 4) had an expression ratio of 4.96 (mean pancreas tumors/mean normals without pancreas) and 4.85 (mean pancreas tumors/mean normals including pancreas). Thus, this cDNA showed over-expression in pancreas tumor tissues as compared to normal pancreas and other normal tissues. In searches against Ganbank, this clone showed some degree of similarity to Human pump-1 mRNA homolog to metalloproteinase, collagenase and stromelysin (Table 4).

Example 3 Analysis of Expression of Pancreatic Tumor cDNA, PN630S, Using Real-time PCR

[0449] Expression patterns of the pancreatic tumor candidate gene, Pn630S (SEQ ID NO:128 and 129), were further analyzed by real-time PCR. The first-strand cDNA to be used in the quantitative real-time PCR was synthesized from 20 μg of total RNA that had been treated with DNase I (Amplification Grade, Gibco BRL Life Technology, Gaitherburg, Md.), using Superscript Reverse Transcriptase (RT) (Gibco BRL Life Technology, Gaitherburg, Md.). Real-time PCR was performed with a GeneAmp™ 5700 sequence detection system (PE Biosystems, Foster City, Calif.). The 5700 system uses SYBR™ green, a fluorescent dye that only intercalates into double stranded DNA, and a set of gene-specific forward and reverse primers. The increase in fluorescence is monitored during the whole amplification process. The optimal concentration of primers was determined using a checkerboard approach and a pool of cDNAs from pancreas tumors was used in this process. The PCR reaction was performed in 25 μl volumes that include 2.5 μl of SYBR green buffer, 2 μl of cDNA template and 2.5 μl each of the forward and reverse primers for the gene of interest. The cDNAs used for RT reactions were diluted 1:10 for each gene of interest and 1:100 for the β-actin control. In order to quantitate the amount of specific cDNA (and hence initial mRNA) in the sample, a standard curve is generated for each run using the plasmid DNA containing the gene of interest. Standard curves were generated using the Ct values determined in the real-time PCR which were related to the initial cDNA concentration used in the assay. Standard dilution ranging from 20-2×10⁶ copies of the gene of interest was used for this purpose. In addition, a standard curve was generated for β-actin ranging from 200fg-2000fg. This enabled standardization of the initial RNA content of a tissue sample to the amount of β-actin for comparison purposes. The mean copy number for each group of tissues tested was normalized to a constant amount of β-actin, allowing the evaluation of the over-expression levels seen with each of the genes.

[0450] Pn630S (SEQ ID NO:128 and 129) was analyzed using an extended panel of pancreas tumor and normal samples. This gene was found to have increased mRNA expression in approximately 70% of pancreas tumors. Elevated expression was also seen in normal pancreas, pancreatitis, breast, gall bladder, kidney and salivary gland. Trace expression was seen in bronchus, skin and uterus. These data indicate that Pn630S may be a valuable as a tumor immunotherapeutic or diagnostic tool.

Example 4 Synthesis of Polypeptides

[0451] Polypeptides are synthesized on a Perkin Elmer/Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence is attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide. Cleavage of the peptides from the solid support is carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides are precipitated in cold methyl-t-butyl-ether. The peptide pellets are then dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) is used to elute the peptides. Following lyophilization of the pure fractions, the peptides are characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

[0452] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 155 <210> SEQ ID NO 1 <211> LENGTH: 719 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 38, 51, 70, 78, 88, 581, 649, 664, 682, 717 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1 attggggttg gggccccaac cacaaatggg gccgaarnat ggsggaaggk ngcaaggtcc 60 tggtgcttkn atgggygnag gcccatyntt cytgggccgc ctggcgggcc atsgtggcta 120 aacaggtact gctgggccgr aaggtggtgg tcgtacgctg tgaaggcatc aacatttctg 180 gcaatttcta cagaaacaag ttgaagtacc tggctttcct ccgcaagcgg atgaacacca 240 acccttcccg aggcccctac cacttccggg cccccagccg catcttctgg cggaccgtgc 300 gaggtatgct gccccacaaa accaagcgag gccaggccgc tytggaccgt ctcaaggtgt 360 ttgacggcat cccaccgccc tacgacaaga aaaagcggat ggtggttcct gctgccctca 420 aggtcgtgcg tctgaagcct acaagaaagt ttgcctatyt ggggcgcctg gctcacgagg 480 ttggctggaa gtaccaggca gtgacagcca ccctggagga gaagaggaaa garaaagcca 540 agatccacta cccggaagaa gaaaacagct catgargctw ncggaaacag gccgagaara 600 acgtggagaa gaaaatttsa caaatacaca gargtcctca agacccacng gaytcytggt 660 ttknagccca ataaaraact gnttawttcc tcaaaaaaaa aaaaaaaaaa aaaaaangg 719 <210> SEQ ID NO 2 <211> LENGTH: 786 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 559, 612, 687, 693, 704, 728, 730, 735, 763, 769 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 2 gatgctctgt gcttagggaa ctgtgagacc cctggagggg tgggtaccgg gaccgcactc 60 agcctggggt ttggaggcgg cctcctatag gaagcgacct gggacctaag atttttagac 120 tgactgtggg ttcactggaa taaaaaggaa gaaacaaaga gcattgcagg catcgggact 180 gtcacatttg acaagatcaa agctgcagga aaatggacag tgaggttcag agagatggaa 240 ggatcttgga tttgattgat gatgcttggc gagaagacaa gctgccttat gaggatgtcg 300 caataccact gaatgagctt cctgaacctg aacaagacaa tggtggcacc acagaatctg 360 tcaaagaaca agaaatgaag tggacagact tagccttaca gtacctccat gagaatgttc 420 cccccattgg aaactgacgc ttggctcctt tcttgtggat ggattttctc aaagtacaca 480 gataaagcat ggtttgtttc agtctccaaa ttcaaacctt tgagtaataa atcacactca 540 aaaatgtaca cccatttant ttgtggtagc aaaatgcaat gcgaaattga atgagaaact 600 gagatttctc antaatggtg aatatttcgc tctttaaacc taaaactctt ctttgagtag 660 cttatatttt gaaccatgaa ttgggtnaaa cantttgcct cttncctctt gaattttgct 720 tttgctgncn aaaantttaa aaccctttcc aactaccttt ttntggggnc cctgtaaaca 780 caaggg 786 <210> SEQ ID NO 3 <211> LENGTH: 566 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 17, 27, 61, 63, 65, 68, 70, 229, 377, 453, 473, 485, 547, 562 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 3 agattttagt tattcangtg ttttcanagt ttcaacaagg gatacagata caagcagctt 60 ntngnggngn ggcaatctgg ggagagaaca tgaaagacac tgtttaacag gcaaataatt 120 ccaggaataa atatacatga atgtgttttt caaaatacag gttcttatac aaatgtataa 180 ctaaatactg attccatagt ggggtggttg taactgaaag ggctttgana aaaggctttg 240 aataaaacta gtcatccacc tagccaaaga tcctttccag cagcacaaaa ggaatttgta 300 aggagaacag agattaactg tcagatatct ttctaatctg taaatttatc caaagtttga 360 aaataccatg aagaatntta ggaatgccag taaccaggga atgggatatt tgcatatcca 420 acatctacag gctaaataca ccccgaatgc ttntcaatgt ggggaaatat ganaagttaa 480 taatnctcct agtgtctggc ttcctttttc tttgcccttt ccctctcata cccttcaatc 540 acctgtntta ccaggacccc cnataa 566 <210> SEQ ID NO 4 <211> LENGTH: 886 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 101, 495, 502, 513, 523, 526, 566, 567, 601, 615, 623, 631, 639, 640, 651, 655, 662, 666, 677, 689, 701, 703, 708, 723, 724, 726, 728, 732, 737, 738, 742, 769, 774, 792, 802, 810, 818, 823, 834, 864, 886 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 4 tctagaacta gtggatccgt tctctgcgat gacataatat gtgacgatca agaattagac 60 tgccccaacc cagaaattcc atttggagaa tgttgtgcag nttgcccaca gcctccaact 120 gctcctactc gccctcctaa tggtcaagga cctcaaggcc ccaagggaga tccaggccct 180 cctggtattc ctgggagaaa tggtgaccct ggtattccag gacaaccagg gtcccctggt 240 tctcctggcc cccctggaat ctgtgaatca tgccctactg gtcctcagaa ctattctccc 300 cagtatgatt catatgatgt caagtctgga gtagcagtag gaggactcgc aggctatcct 360 ggaccagctg gccccccagg ccctcccggt ccccctggta catctggtca tcctggttcc 420 cctggatctc caggatacca aggaccccct ggtgaacctg gggcaagctg ggtcctttaa 480 ggccttccca ggaancttcc tnggggctat tanggtccat ctnggncctt gcttggaaaa 540 aagaatgggg aagaaaatca agggtnngaa cccccggaac caaaaccttg ggaaaaaacc 600 naaagggaat ttggnccttg ggnaaccctt ncccagggnn ttttcaaaaa ngggnccccc 660 ancctngggg gaaaaanccc ttggggaant tccccttggg ntntttgnaa aaaggggccc 720 ccnngnangg gnttttnnaa tngggaccca aaaaatgggg agaaaaaang gggnggaaaa 780 caagggggct tncttggaat tnaaaggggn gaaaaaangg gcntttccag ggcnaaaaat 840 ggagcttctt ggaacccttg ggtnccaaaa aggggttctt ggggan 886 <210> SEQ ID NO 5 <211> LENGTH: 780 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 456, 489, 532, 562, 608, 622, 628, 640, 659, 672, 675, 701, 704, 712, 718, 772, 779 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 5 accgtcatta ccccgagcac ctgcagcccc aggaagtcct ggccgtcctc gctcaccagg 60 agcccctctt ggacccatgg gtccaggagc tccattttcg cctggaagac cattttcacc 120 ctttaatcca ggagcacctg tttcaccctt ttctccattt cgtccatcga agcctctgtg 180 tcctttcata ccagggaatc caggtatccc agctggacct ttgatacctg gaggtccagg 240 caatcctcgc tctccaggtc gtccgggtct acctgattct ccatcttttc cagcaggacc 300 agatggacct atagcaccag gaggtcctgg agggcctgaa ggaccagctt gcccaggttc 360 accagggggt ccttggtatc ctggagatcc aggggaacca ggatgaccag atgtaccagg 420 gggaccggga gggcctgggg gggccagctg gtccangata gcctgcgagt cctcctactg 480 ctacttcana cttgacatca tatgaatcat actggggaga ataagttctg angaccagta 540 gggcatgatt cacagattcc angggggcca ggagaaccag gggaccctgg gttgtcctgg 600 aataccangg tcaccatttc tnccaagnaa tacccaggan gggctgggat cttcccttng 660 ggggcctttg anggnccctt gaccatttag gaaggggcaa ntanggaacc anttgggngg 720 ggttgggggg caaaactgca caaaattttt tccaaaggga attttttggg gnggggggng 780 <210> SEQ ID NO 6 <211> LENGTH: 693 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 361, 370, 388, 465, 466, 468, 474, 476, 480, 483, 500, 517, 541, 547, 557, 570, 574, 575, 585, 587, 607, 616, 627, 635, 643, 674, 692 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 6 tcgagagaga gaccgagagg ttcagggagc tgttgctgta tggccgtaag aaggatgctt 60 tggagtctgc aatgaagaat ggcctgtggg gtcacgctct gctacttgcr rgtargatgg 120 acagccggac acacgcccga gtcatgacca ggtttgctaa cagcctccca atcaacgacc 180 ctctgcagac agtctaccag ctcatgtccg gacggatgcc tgccgcgtcc acgtgctgtg 240 gagacgagaa atggggagat tggaggccgc acctcrccat ggtcttgkcc aacttgaaca 300 acaacatgga crtssagtyc asracsatgg ytaycatkgg cgacactstg gcktyraggg 360 ncctacyygn awgyrgccca mttytgcnta cctmatgscc caggcggrat ttggtgttta 420 cackaakaaa wctacaaakc ttgtcttaat cggatcaact tcttnnanac tcgncncccn 480 acnggggggt ggaactccan cttttgttcc cttttantga ggggttaatt tgcgccgctt 540 nggcgtnaat catgggncat agctgttacn ttgnngaaaa attgntnatc cgcttacaat 600 tccacancaa cattanaacc cggaaancat taaantggta aangcctggg ggtgcctaaa 660 ttgaagtgga gctnaccttc ccctttaatt tnc 693 <210> SEQ ID NO 7 <211> LENGTH: 623 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 48, 50, 53, 529, 565, 569, 598, 616 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 7 gggaagtgac atcgtcttta aaccctgcgt ggcaatccct gacgcacngn cgngatgccc 60 agggaagaca gggcgacctg gaagtccaac tacttcctta agatcatcca actattggat 120 gattatccga aatgtttcat tgtgggagca gacaatgtgg gctccaagca gatgcagcag 180 atccgcatgt cccttcgtgg gaaggctgtg gtgctgatgg gcaagaacac catgatgcgc 240 aaggccatcc gagggcacct ggaaaacaac ccagctctgg agaaactgct gcctcatatc 300 cgggggaatg tgggctttgt gttcaccaag gaggacctca ctgagatcag ggacatgttg 360 ctggccaata aggtgccagc tgctgcccgt gctggtgcca ttgccccatg tgaagtcact 420 gtgccagccc agaacactgg tctcgggccc gagaagactc ctttttccag gctttaggta 480 tcaccactaa aatctccagg ggcaccattg aaatctgagt gatgtgcanc tgatcaagac 540 tggagacaag tgggagccag cgaancccnc ttcttaacat gctcaacatc tccccttntc 600 ctttgggctg gtcatncagc agg 623 <210> SEQ ID NO 8 <211> LENGTH: 544 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 4, 21, 65, 116, 234, 309, 363, 392, 444, 450, 486, 489, 506, 522 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 8 tttntttttt tttttttaaa naagtaagcc tttatttcct tgttttgcaa ataaaactgg 60 ctaanttggt tgctttttgg tgattaatca aagggggggg gggccatatc ctcgtncgac 120 tcctccgact cttccttggc ttcaacctta gctggggctg cagcagcagc aggagcagct 180 gtggtggcag cagccacagg ggcagcagcc acaaaggcag atggatcagc caanaaggcc 240 ttgacctttt cagcaagtgg gaaggtgtaa tccgtctcca cagacaaggc caggactcgt 300 ttgtacccnt tgatgataga atggggtact gatgcaacag ttgggtagcc aatctgcaga 360 canacactgg caacattgcg gacaccctcc angaagcgag aatgcagaat ttcctctgtg 420 atatcaagca cttcagggtt gtanatgctn ccattgtcca acacctgctg gatgaccagc 480 ccaaangana aaggggagat ttttancatt ttcaacaacg gngcttccct tgcttcccac 540 tttt 544 <210> SEQ ID NO 9 <211> LENGTH: 92 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 attttcaaat gttaaaccat gtattagtcc attttcacac tgctaataaa gacatacatc 60 agactgggca atttccaaaa aaaaaaaaaa aa 92 <210> SEQ ID NO 10 <211> LENGTH: 748 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 83, 528, 539, 572, 591, 607, 620, 623, 649, 654, 665, 670, 686, 737, 745 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 10 tgggcgacgt cttcatcggc cgctactaca ctgtgtttga ccgtgacaac aacagggtgg 60 gcttcgccga ggctgcccgc ctntagttcc caaggcgtcc gcgcgccagc acagaaacag 120 aggagagtcc cagagcagga ggcccctggc ccagcggccc ctcccacaca cacccacaca 180 ctcgcccgcc cactgtcctg ggcgccctgg aagccggcgg cccaagcccg acttgctgtt 240 ttgttctgtg gttttcccct ccctgggttc agaaatgctg cctgcctgtc tgtctctcca 300 tctgtttggt gggggtagag ctgatccaga gcacagatct gtttcgtgca ttggaagacc 360 ccacccaagc ttggcagccg agctcgtgta tcctggggct cccttcatct ccagggagtc 420 ccctccccgg ccctaccagc gcccgctggg ctgagcccct accccacacc aggccgtcct 480 cccgggccct cccttggaaa cctgccctgc tgagggcccc tctgccanct tgggcccanc 540 tgggcttttg caccctactg ttcaatgtcc cnggcccctt gaggatgaag nccgctagaa 600 gcctgangat aacttggaan gantgaaaag ggacaaaaac cccccttgnt gganccttga 660 agggnggtgn ttgggactta acccantccc aaggggcatg tattgggcct tgaagggggg 720 ggttgggaat tgggggnttg gggcnacc 748 <210> SEQ ID NO 11 <211> LENGTH: 453 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1, 14, 92, 105, 115, 139, 165, 175, 176, 183, 186, 188, 190, 192, 195, 196, 199, 202, 207, 208, 210, 219, 224, 232, 238, 243, 247, 281, 285, 303, 305, 307, 308, 314, 315, 319, 320, 360, 384, 391, 394, 400, 412, 429 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 11 ngacgaaggc tggnaccacc ccccaatccc aaccccacct ccaggccaat acatgcccct 60 gggactggct cagttccaac accaccctgc angctccaac aaggngggtt ttgtnccctc 120 tcactccttc cagctcatnc tcaggtctct agcgggctca tcctnaacgg gcccnntaca 180 ctnaanangn anggnngcnt ancccanntn ggcccaatnt gggnagaggg gncctcangc 240 agngcangtt tccaatggag ggcccgggag gaccgcctga ngtgnggtaa gggatcagcc 300 canangnncg cttnntacnn gcctgggaag gggactccct ttaacatgaa cgtaaccccn 360 cgatatacga actcccctgc caanattggg nggngtcttn caatgcctaa anagaaaatc 420 tctcttganc aactctcccc accaaacata tgg 453 <210> SEQ ID NO 12 <211> LENGTH: 738 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 25, 28, 51, 62, 65, 138, 166, 199, 225, 246, 294, 376, 387, 404, 419, 437, 438, 453, 471, 472, 489, 493, 501, 505, 515, 529, 548, 570, 578, 585, 592, 596, 605, 608, 609, 622, 626, 633, 641, 658, 666, 731, 732 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 12 gccatgggcc gccgccccgc ccgtnggnac cggtattgta agaacaagcc ntacccaaag 60 tntcncttct gccgaggtgt ccctgatgcc aagattcgca tttttgacct ggggcggaaa 120 aaggcaaaag tggatgantt tccgctttgt ggccacatgg tgtcanatga atatgagcag 180 ctgtcctctg aagccctgna ggctgcccga atttgtgcca ataantacat ggtaaaaagt 240 tgtggnaaag atggcttcca tatccgggtg cggctccacc ccttccacgt catncgcatc 300 aacaagatgt tgtcctgtgc tggggctgac aggctccaaa caggcatgcg aggtgccttt 360 ggaaagcccc acggcnctgt ggccagngtt cacattggcc aagntatcat gtccatccnc 420 accaagctgc aaaacannga gcatgtgatt gangccctgc gcaaggccaa nntcaaagtt 480 tcctggccnc canaaaaatc ncatnttaaa gaagnggggc ttcacccant tcaatgctga 540 tgaaattnaa gacatgtggc tgaaaaacgn ctatccanat ggtgnggggg tnaaanacat 600 cccantcnng cctcttgaca anggcnggcc ctncactcat naggctttca atgggctncc 660 cccctnttaa actcacaata aaatttcctt ctttccccta aaaaaaaaaa aaaaaaaagg 720 ggggccgttc nngggggg 738 <210> SEQ ID NO 13 <211> LENGTH: 546 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 cattcgcagc ctttagcatc atgtagaagc aaactgcacc tatggctgag ataggtgcaa 60 tgacctacaa gattttgtgt tttctagctg tccaggaaaa gccatcttca gtcttgctga 120 cagtcaaaga gcaagtgaaa ccatttccag cctaaactac ataaaagcag ccgaaccaat 180 gattaaagac ctctaaggct ccataatcat cattaaatat gcccaaactc attgtgactt 240 tttattttat atacaggatt aaaatcaaca ttaaatcatc ttatttacat ggccatcggt 300 gctgaaattg agcattttaa atagtacagt aggctggtat acattaggaa atggactgca 360 ctggaggcaa atagaaaact aaagaaatta gataggctgg aaatgcttac tttctgctct 420 cattttcttt cattttgtct sctgtctttt tgaatgccaa tactttcatt taaaatgttt 480 tgggagcacg gcaacccaaa atgactgaac ggtggtccaa aaatccaaac caactgataa 540 ttactt 546 <210> SEQ ID NO 14 <211> LENGTH: 827 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11, 13, 61, 62, 82, 85, 90, 706, 776, 817 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 14 cggccaaggg ngncaattta ccccttcatt taaagggaac caaaaggtgg gggctccccc 60 nncggtggcg gcccgctttt anaanctagn ggttccrggk tatccccaaa atgatagcac 120 atgactttcc tggaattggc cacaaagttg atgcagtttt catgaaagat ggatttttct 180 atttctttca tgggaacaag accaatacaa atttgatcct aaaacgaaga gaattttgac 240 tctccagaaa gctaatagct ggttcaactg caggaaaaat tgaacattac taatttgaat 300 ggaaaacaca tggtgtgagt ccaaaggagg tgttttcctg aagaactgtc tattttctca 360 gtcattttta acctctagag tcactgatac acagaatata atcttattta tacctcagtt 420 tgcatatttt tttactattt agaatgtagc cctttttgta ctgatataat ttagttccac 480 aaatggtggg tacaaaaagt caagtttgtg gcttatggat tcatataggc cagagttgca 540 aagatctttt ctagagtatg caactctgac gttgatccca gagagcagct tcagtgacaa 600 acatatcctt tcaagacaga aagagacagg agacatgagt ctttgccgga ggaaaagcag 660 ctcaagaaca catgtgcagt cactggtgtc accctggata ggcaanggsa taactcttct 720 aacacaaaat aaggtgkttt atgtttggaa taaagtcaac ccttgkttct actgknttta 780 tacactttct ataaaaaaaa aaaaaaaaaa gggcggncgc tccaagg 827 <210> SEQ ID NO 15 <211> LENGTH: 232 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 109, 127, 151, 153, 180, 217, 219 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 15 gtaacttcgg gataaggatt ggctctaagg gctgggtcgg tcgggctggg gcgcgaagcg 60 gggctgggcg cgcgccgcgg ctggacgagg cgccgccgcc ccccccacnc ccggggcacc 120 cccctcncga aacctccccc gccccacccc ncncacgccg ctcactccct ccccgccccn 180 cgccctctct ctctctctct ctctcccccg ctccccntnc tcccccctcc cc 232 <210> SEQ ID NO 16 <211> LENGTH: 211 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 gcagttccct cctccttgtg gccgttgcct caggctatgg cccaccttcc tctcactctt 60 ccagccgcgt tgtccatggt gaggatgcgg tcccctacag ctggccctgg caggtttccc 120 tgcagtatga gaaaagtgga agcttcatac cacacgtgtg gcggtagcct catcgccccc 180 gattgggttg tgactgccgg ccactgcatc t 211 <210> SEQ ID NO 17 <211> LENGTH: 652 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 3, 7, 409, 457, 460, 472, 476, 478, 483, 485, 502, 506, 510, 517, 520, 523, 533, 534, 539, 576, 592, 598, 623, 632 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 17 ggncctncta atgacctccg gcctagccat gtgatttcac ttccactcca taacgctcct 60 catactaggc ctactaacca acacactaac catataccaa tgatggcgcg atgtaacacg 120 agaaagcaca taccaaggcc accacacacc acctgtccaa aaaggccttc gatacgggat 180 aatcctattt attacctcag aagttttttt cttcgcagga tttttctgag ccttttacca 240 ctccagccta gcccctaccc cccaattagg agggcactgg cccccaacag gcatcacccc 300 gctaaatccc ctagaagtcc cactcctaaa cacatccgta ttactcgcat caggagtatc 360 aatcacctga gctcaccata gtctaataga aaacaaccga aaccaaatna ttcaagcact 420 ggttattaca attttactgg ggctctattt taccctnctn caagcctcag angacntnga 480 atntnccttt accattttcg anaggntttn acgggtnaan atntttttgg tanncaggnt 540 tccaccggac ttcacgtatt attggggtca actttnctca ctatctgggt tnatccgnca 600 actaaatatt ttactttaca ttncaaaaaa tnacttttgg ggttttaaaa ac 652 <210> SEQ ID NO 18 <211> LENGTH: 1089 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 682, 759, 770, 783, 784, 787, 790, 809, 811, 817, 822, 825, 841, 852, 854, 867, 869, 873, 884, 885, 890, 891, 892, 895, 912, 915, 916, 917, 922, 924, 925, 927, 928, 935, 936, 943, 944, 945, 954, 960, 971, 985, 988, 1007, 1021, 1022 <223> OTHER INFORMATION: n = A,T,C or G <221> NAME/KEY: misc_feature <222> LOCATION: 1030, 1078, 1089 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 18 tttccggccg cgccccgttt cccaggacga agggcactcc gcaccggacc ccggtcccgg 60 cgcgcggcgg ggcacgcgcc ctcccgcgcg cgcggggcgc gtggaggggg ggggcaggcc 120 cgccggcggg gacaggcggg ggaccggcta tccgaggcca accgaggctc cgcggcgctg 180 ccgtatcgtt ccgcctgggc gggattctga cttagaggcg ttcagtcata atcccacaga 240 tggtagcttc gccccattgg ctcctcagcc aagcacatac accaaatgtc tgaacctgcg 300 gttcctctcg tactgagcag gattaccatg gcaacaacac atcatcagta gggtaaaact 360 aacctgtctc acgacggtct aatcccagct cacgttccct attagtgggt gaacaatcca 420 acgcttggtg aattctgctt cacaatgata ggaagagccg acatcgaagg atcaaaaagc 480 gacgtcgcta tgaacgcttg gccgccacaa gccagttatc cctgtggtaa cttttctgac 540 acctcctgct taaaacccaa aaggtcagaa ggatcgtgag gccccgcttt cacggtctgt 600 attcgtactg aaaatcaaga tcaagcgagc ttttgccctt ctgctccacg ggaggtttct 660 tgtcctccct gagctcgctt angacacctg cgttaccgtt tgacaaggtg tacccgcccc 720 aatcaaaact tcccccacct ggcacttgtc ccccggaanc ggggtcgcgn ccccggcccg 780 ggnnccnccn ggccggggcc gctttgggnc nccccanaaa anccnaaaaa accccccttc 840 nggggggttt tngncccccc ccccggncnt ttnaacccgg gggnntcaan nnggnaaaaa 900 aaaaacccaa tnccnnnaaa antnngnngg gggtnntttt ttnnnccccg gggngggggn 960 ccccccccaa ngggggcccc ggggnggnaa accccccccc ccccccnggg ggcccccttt 1020 nngggggggn acccccccgg gggggggggc gccccggggg gccttccccc tttttttnac 1080 ccttttatn 1089 <210> SEQ ID NO 19 <211> LENGTH: 231 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 ataaaacctc aagctatgga ataccctgag tgtgtgcaaa tacactgtcc aggatgagag 60 ccactcagag tgggtgtctt gtgtccgctt ctcgcccaac agcggcaacc ctatcatcgt 120 ctcctgtggc tgggacaagc tggtcaaggt atggaacctg gctaactgca agctgaagac 180 caaccacatt ggccacacag gctatctgaa cacggtgact gtctctccag a 231 <210> SEQ ID NO 20 <211> LENGTH: 714 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 82, 651, 684, 705 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 20 ctgcatcctc aacaacaatg tgcagtgagc atgtggaaga aaagaagcag ctttacctac 60 ttgtttcttt ttgtctctct tnctggacac tcactttttc agagactcaa cagtctctgc 120 aatggagtgt gggtccacct tagcctctga cttcctaatg taggaggtgg tcagcaggca 180 atctcctggg ccttaaagga tgcggactca tcctcagcca gcgcccatgt tgtgatacag 240 gggtgtttgt tggatgggtt taaaaataac tagaaaaact caggcccatc cattttctca 300 gatctccttg aaaattgagg ccttttcgat agtttcgggt caggtaaaaa tggcctcctg 360 gcgtaagctt ttcaaggttt tttggaggct ttttgtaaat tgtgatagga actttggacc 420 ttgaacttac gtatcatgtg gagaagagcc aatttaacaa actaggaaga tgaaaaggga 480 aattgtggcc aaaactttgg gaaaaggagg ttcttaaaat cagtgtttcc cctttgtgca 540 cttgtagaaa aaaaagaaaa accttctaag ctgatttgat ggacaatgga gagagcttcc 600 ctgtgattat aaaaaaggaa gctagctgct ctacgggcat ctttgcttaa nagtatactt 660 taacctggct ttttaaagca gtangtaact gccccaccca aaggncttta aaaa 714 <210> SEQ ID NO 21 <211> LENGTH: 572 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 gcatgaatct acttctgatc cttacctttg ttgcagctgc tgttgctgcc ccctttgatg 60 atgatgacaa gatcgttggg ggctacatct gtgaggagaa ttctgtcccc taccaggtgt 120 ccttgaattc tggctaccac ttctgcggtg gctccctcat cagcgaacag tgggtggtgt 180 cagcaggtca ctgctacaag tcccgcatcc aggtgagact gggagagcac aacatcgaag 240 tcctggaggg gaatgaacag ttcatcaatg cagccaagat catccgccac cccaaataca 300 acagccggac tctggacaat gacatcctgc tgatcaagct ctcctcacct gccgtcatca 360 attcccgcgt gtccgccatc tctctgccca ctgcccctcc agctgctggc accgagtccc 420 tcatctccgg ctggggcaac actctgagtt ctggtgccga ctacccagac gagctgcagt 480 gcctggatgc tcctgtgctg agccaggctg agtgtgaagc ctcctaccct ggaaagatta 540 ccaacaacat gttctgtgtg ggcttcctcg ag 572 <210> SEQ ID NO 22 <211> LENGTH: 963 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 26, 39, 40, 43, 44, 46, 54, 57, 948, 956 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 22 ccttttttga aaaaaaaaaa aaattntttt ttttttttnn ccnntncccg gggnccnttt 60 ttcccccctc ccttcccaga ttccgtctat gtccacaatc tgagagagaa tgttcttttt 120 acatccaggc atttccactc ctccatttgg aaagaaatct aggtggccca cggagttggg 180 ctcattccaa accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca 240 aatttggcat cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct 300 gctgggtcca accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca 360 gcagcgtggg cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac 420 gccgactgaa gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc 480 gaggcttgtg tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca 540 ctttccacct tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct 600 atgaatccat gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg 660 cttgatgaat ctgcggcaac ttcttgaaag ttgtttgggt tctcattagt atataggagg 720 aagcgggtgt tgacatcttt tggagaccaa ggcaatatat ggaggggtct ttccgtaatt 780 cctgaccatg ggggagtcat cactgaagca gccgagtctt tcgtagcaaa cttcttttcc 840 tgctactgct cccascagca gtgaaagagt ccaaagtggc agcatcgtgg cagttccgtc 900 cggycmcmmy tgggmwakks smycccyymt tkggacttag gggggaancc ccctantttc 960 tta 963 <210> SEQ ID NO 23 <211> LENGTH: 412 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 206, 247, 254, 257, 290, 299, 312, 340, 347, 355, 383, 386 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 23 gcccagatag gggagcggag gtggcggcgg cggcggtagc ggtggccttg gttgtcttcc 60 agtctcctcg gctcgccctt tagccggcac cgctcccctt ccctccccct tcctctcttc 120 cttccttccc tccccttccc tttttccctt ccccgtcggt gagcggcggg ggtggctcca 180 gcaacggctg ggcccaacct gtgtanaggc cttaaccaac gataacggcg gcgacggcga 240 aacctcngag ctcncanggc gggggcaaag cccgggcctt ggagatagan aattctcant 300 tgtgtaagct gntcatcggc ggcctcaatg tgcagactan tgagtcnggc ctgcncggcc 360 actttgacgc ctttgggact ctnacngact gcatggtggt ggtgaatccc ca 412 <210> SEQ ID NO 24 <211> LENGTH: 232 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 74, 78, 209, 222 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 24 cctggtgctg atggtcctgc tggggctcct ggtactcccg ggcctcaagg tattgctgga 60 cagcgtggtg tggncggnct gcctggtcag agaggagaga gaggcttccc tggtcttcct 120 ggcccctctg gtgaacctgg caaacaaggt ccctctggag caagtggtga acgtggtccc 180 cctggtccca tgggcccccc tggattggnt ggaccccctg gngaatctgg ac 232 <210> SEQ ID NO 25 <211> LENGTH: 472 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 445, 472 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 25 ggacgggcgc gtcttcaggg tggaagcctg gcgcacgtcc ggaggtgccg aggacccaac 60 cagcccaaac tctgggggaa atgactcccc tctgccctcg ccccgcgctc tgctaccatt 120 tccttacgtc tctgcttcgc tcagcgatgc aaaacgcgcg aggcgcacgg cagagggccg 180 aagccgcggt actctccggg ccaggcccgc ccctcggccg cgccgcgcag cacgggattc 240 cccggccgct gtccagcgct ggccgcctga gccaaggctg ccgcggagcc agtacagtcg 300 gggccgctgg ctggaagggc gagcttccta aggcgggggg aagcccggcg ccggggccgg 360 gtaggaaagg cgggggaggg gctccggccg tctggaagga atccacgcgg cttgaggctg 420 tgggggaagt agggtggcga gcggnccttc tgcgcgcggg gggcgggggg gn 472 <210> SEQ ID NO 26 <211> LENGTH: 219 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 129, 135, 138, 140, 154, 163, 166, 181, 206 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 26 gtaacttcgg gataaggatt ggctctaagg gctgggtcgg tcgggctggg gcgcgaagcg 60 gggctgggcg cgcgccgcgg ctggacgagg cgccgccgcc ccccccacgc ccggggcacc 120 cccctcgcng tcctnccncn ccccaccccg cgcnctcaat tcnctncctc cccacccccc 180 nccctctatc tctctctctc ccccantacc cctcctccc 219 <210> SEQ ID NO 27 <211> LENGTH: 546 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 27 aagtaattat nagttggttt ggatttttgg accaccgttc agtcattttg ggttgccgtg 60 ctcccaaaac attttaaatg aaagtattgg cattcaaaaa gacagcagac aaaatgaaag 120 aaaatgagag cagaaagtaa gcatttccag cctatctaat ttctttagtt ttctatttgc 180 ctccagtgca gtccatttcc taatgtatac cagcctactg tactatttaa aatgctcaat 240 ttcagcaccg atggccatgt aaataagatg atttaatgtt gattttaatc ctgtatataa 300 aataaaaagt cacaatgagt ttgggcatat ttaatgatga ttatggagcc ttagaggtct 360 ttaatcattg gttcggctgc ttttatgtag tttaggctgg aaatggtttc acttgctctt 420 tgactgtcag caagactgaa gatggctttt cctggacagc tagaaaacac aaaatcttgt 480 aggtcattgc acctatctca gccataggtg cagtttgctt ctacatgatg ctaaaggctg 540 cgaatg 546 <210> SEQ ID NO 28 <211> LENGTH: 823 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 ggatccgttc tctgcgatga cataatatgt gmcgatcaag aaattagmct gccccaaccc 60 agaaattcca tttggagaat gttgtgcagt ttgccccaca gcctccaact gctcctactc 120 gccctcctaa tggtcaagga cctcaaggcc ccaagggaga tccaggccct ccttggtatt 180 cctgggagaa atggtgaccc tggtattcca ggacaaccag ggtcccctgg ttctcctggc 240 ccccctggaa tctgtgaatc atgccctact ggtcctcaga actattctcc ccagtatgat 300 tcatatgatg tcaagtctgg agtagcagta ggaggactcg caggctatcc tggaccagct 360 ggccccccag gccctcccgg tccccctggt acatctggtc atcctggttc ccctggatct 420 ccaggatacc aaggaccccc tggtgaacct gggcaagctg gtccttcagg ccctccagga 480 cctcctggtg ctataggtcc atctggtcct gctggaaaag atggagaatc aggtagaccc 540 ggacgacctg gagagcgagg attgcctgga cctccaggta tcaaaggtcc agctgggata 600 cctggattcc ctggtatgaa aggacacaga ggcttcgatg gacgaaatgg agaaaagggg 660 tgaaacaggt gctcctggat taaagggtga aaatggtctt ccaggcgaaa aatggagctc 720 ctggacccat gggtccaaga rgggctcctg gtgagcgagg acggccagga cttcctgggg 780 ctgcaggtgc tcggggtaat gacggtgctc gagggggggc ccg 823 <210> SEQ ID NO 29 <211> LENGTH: 824 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 691, 785, 816 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 29 cgctggtcat gaagcagtgc cacccggaca ccaagaagtt cctgtgctcg ctcttcgccc 60 ccgtctgcct cgatgaccta gacgagacca tccagccatg ccactcgctc tgcgtgcagg 120 tgaaggaccg ctgcgccccg gtcatgtccg ccttcggctt cccctggccc gacatgcttg 180 agtgcgaccg tttcccccag gacaacgacc tttgcatccc cctcgctagc agcgaccacc 240 tcctgccagc caccgaggaa gctccaaagg tatgtgaagc ctgcaaaaat aaaaatgatg 300 atgacaacga cataatggaa acgctttgta aaaatgattt tgcactgaaa ataaaagtga 360 aggagataac ctacatcaac cgagatacca aaatcatcct ggagaccaag agcaagacca 420 tttacaagct gaacggtgtg tccgaaaggg acctgaagaa atcggtgctg tggctcaaag 480 acagcttgca gtgcacctgt gaggagatga acgacatcaa cgcgccctat ctggtcatgg 540 gacagaaaca gggtggggag ctggtgatca cctcggtgaa gcggtggcag aaggggcaga 600 gagagttcaa gcgcatctcc cgcagcatcc gcaagctgca gtgctagtcc ccggcatcct 660 gatggctccg acaggcctgg ctccagacac nggttgacca tttctggttc cggggatctc 720 agcttcccgt tcccccaagc acacttcctt agcttggctt ccagttcttc aagcctgggg 780 caggntttcc cccttgcctt tttgcacgtt ttggcnttcc ccca 824 <210> SEQ ID NO 30 <211> LENGTH: 821 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 713, 730, 821 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 30 tttttttttt tttttaggta aaacaggatg taaagtttat atacaagaat ataatgttta 60 tctgaaatat ttacagtgtt ggttaaagca atatttttac aacttttaaa ggtaaactac 120 tatgtatatt acaggtaagc tacaatgggt ttaatttgca aaagttaagt aagaaatgtt 180 ttaaacaagg cttaaagtac tcaagtcaat tataaaattt atatcttttg ccttttactt 240 gaagaaatca tgctatagaa atggttaatg tgcttctaat aaatggaagt attgtagctg 300 gaatgtgata catgtaacag tttaagttcc cattgaaggt ataaaatgat gaattgttgt 360 aagacttaga cactgagtct cagtctggag ctgatgaaga tgttgagata acagccagct 420 ttatctcaac agggtttgtg acccacaagt ttgggccaca gagaaaattg aagcaatttg 480 catgttatga caacctcagt gggaagtgaa aatcagctga ctcaaaacaa caaacaacaa 540 ccaaccagac ccaagtcaca gttgcaccta ttcaaaacta gctttaaagt gagctatttt 600 taaacttcat aaaaatattc atgattttat tagtttgaat atttctacaa gattcgggtg 660 ggcttttcct ttaggtgaaa acagctatcc actcctgtgg ccttataact cangaaatgc 720 tggggatgcn aacgtgcaaa aggcaggggg aagctgccca gctgagactg gacagctagg 780 agtgtgcttg ggggaacggg gagctgagaa ccccgggaca n 821 <210> SEQ ID NO 31 <211> LENGTH: 588 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31 gctggccgam tgcacctggc acatggcgga gaagytgggc tgcttctggc ccaatgcaga 60 ggtggacagg ttcttcctgg cagtgcatgg ccgctacttc aggagctgcc ccatytcagg 120 cagggccgtg cgggacccgc ccggcagcat cctctacccc ttcatcgtgg tccccatcac 180 ggtgaccctg ctggtgacgg cactggtggt ctggcagagc aagcgcactg agggcattgt 240 gtaggcgggg cccaggctgc ccgcgactgc acccaggctg cagggtgagg ccaggcaggc 300 ctgggtaggg gcagcttytg gagccttggg acagagcagg cccacaatgc cccccttctt 360 ccagccaaga agagctcaca ggagtccaga gtagccgagg ctctggtatt aacctggaag 420 cccccctggc tggaggccac cgccacccta ggaagggggc agggacgtga ccttgactta 480 cctctggaaa gggtcccagc ctagactgct taccccatag ccacatttgt ggatgagtgg 540 tttgtgatta aaagggatgt tcttgaaaaa aaaaaaaaaa aaaaaaag 588 <210> SEQ ID NO 32 <211> LENGTH: 594 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 528, 574, 594 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 32 ctgtttgtgg gtgtggacac catccacgtc cacaagagat ggaatgccct cctgttgcgc 60 aatgatattg ccctcatcaa gcttgcagag catgtggagc tgagtgacac catccaggtg 120 gcctgcctgc cagagaagga ctccctgctc cccaaggact acccctgcta tgtcaccggc 180 tggggccgcc tctggaccaa cggccccatt gctgataagc tgcagcaggg cctgcagccc 240 gtggtggatc acgccacgtg ctccaggatt gactggtggg gcttcagggt gaagaaaacc 300 atggtgtgcg ctgggggcga tggcgtcatc tcagcctgca atggggactc cggtggccca 360 ctgaactgcc agttggagaa cggttcctgg gaggtgtttg gcatcgtcag ctttggctcc 420 cggcggggct gcaacacccg caagaagccg gtagtctaca cccgggtgtc cgcctacatc 480 gactggatca acgagaaaat gcagctgtga tttgttgctg ggagcggngg cagcgagtcc 540 ctgcaacagc aataaacttc cttctcctcg ggcnaaaaaa aaaaaaaaag ggcn 594 <210> SEQ ID NO 33 <211> LENGTH: 117 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 33 catggggagt catcactgaa gcagccgagt ctttcgtagc aaacttcttt tcctgctact 60 gctcccagca gcagtgaaag agtccaaagt ggcagcatcg tggcagttcc gtcaggc 117 <210> SEQ ID NO 34 <211> LENGTH: 534 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 514, 520 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 34 ttcgtgtcct ccctggtgaa gagcattggt aacagctact catacgtctg gattgggctc 60 catgacccca cacagggcac cgagcccaat ggagaaggtt gggagtggag tagcagtgat 120 gtgatgaatt actttgcatg ggagagaaat ccctccacca tctcaagccc cggccactgt 180 gcgagcctgt cgagaagcac agcatttctg aggtggaaag attataactg taatgtgagg 240 ttaccctatg tctgcaagtt cactgactag tgcaggaggg aagtcagcag cctgtgtttg 300 gtgtgcaact catcatgggc atgagaccag tgtgaggact caccctggaa gagaatattc 360 gcttaattcc cccaacctga ccacctcatt cttatctttc ttctgtttct tcctccccgc 420 tgtcatttca gtctcttcat tttgtcatac ggcctaaggc tttaaagagc aataaaattt 480 ttagtctgca aaaaaaaaaa aaaaaaaggg cggncgctcn aggggggggg ccgg 534 <210> SEQ ID NO 35 <211> LENGTH: 206 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 35 agtgggttac acaagctacg gcactttgga cagcaaataa gattgtttct gattatggaa 60 aggacccatc catcacttcc attctggacg ccctggatat cttcctcctg ccagtcacaa 120 accctgatgg atacgtgttc tctcaaacca aaaatcgtat gtggcggaag acccggtcca 180 aggtatctgg aagcctctgt gttggt 206 <210> SEQ ID NO 36 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 36 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt cctggagcag cagaataaga tcctgctggc cga 533 <210> SEQ ID NO 37 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 37 ggaacaattg tckctggacg gcagctatgc gactcaccgt gctgtgtgct gtgtgcctgc 60 tgcctggcag cctggccctg ccgctgcctc aggaggcggg aggcatgagt gagctacagt 120 gggaacaggc tcaggactat ctcaagagat tttatctcta tgactcagaa acaaaaaatg 180 ccaacagttt agaagccaaa ctcaaggaga tgcaaaaatt ctttggccta cctataactg 240 gaatgttaaa ctcccgcgtc atagaaataa tgcagaagcc cagatgtgga gtgccagatg 300 ttgcagaata ctcactattt ccaaatagcc caaaatggac ttccaaagtg gtcacctaca 360 ggatcgtatc atata 375 <210> SEQ ID NO 38 <211> LENGTH: 235 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 38 gcacaaaact catgaggctc cggctgctca gttccctcct ccttgtggcc gttgcctcag 60 gctatggccc accttcctct cgcccttcca gccgcgttgt caatggtgag gatgcggtcc 120 cctacagctg gccctggcag gtttccctgc agtatgagaa aagtggaagc ttctaccaca 180 cgtgtggcgg tagcctcatc gcccccgatt gggttgtgac tgccggccac tgcat 235 <210> SEQ ID NO 39 <211> LENGTH: 636 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 550, 613, 636 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 39 ggcttccaac atgtggcagg ctctgggcct ccctctgctg cctgctggtg ttggccaatg 60 cccggagcag gccctctttc catcccctgt cggatgagct ggtcaactat gtcaacaaac 120 ggaataccac gtggcaggcc gggcacaact tctacaacgt ggacatgagc tacttgaaga 180 ggctatgtgg taccttcctg ggtgggccca agccacccca gagagttatg tttaccgagg 240 acctgaagct gcctgcaagc ttcgatgcac gggaacaatg gccacagtgt cccaccatca 300 aagagatcag agaccagggc tcctgtggct cctgctgggc cttcggggct gtggaagcca 360 tctctgaccg gatctgcatc cacaccaatg cgcacgtcag cgtggaggtg tcggcggagg 420 acctgctcac ctgctgtggc agcatgtgtg gggacggctg taatggtggc tatcctgctg 480 aagcttggaa cttctggaca agaaaaggcc tggtttctgg tggcctctat gaatcccatg 540 tagggtgcan accgtactcc atccctccct gtgagcacca cgtcaacggc tcccggcccc 600 atgcacgggg ganggagata cccccaagtg tacaan 636 <210> SEQ ID NO 40 <211> LENGTH: 395 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 333, 338, 359, 388 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 40 gcccaacaca atgggtattc ctagccctgg acaccttcta tggggtgggc cgtgggatga 60 gggacagagg aggaggccgc ctagagccat ccttcaggcc cttgctctgc caccgcctgt 120 tacccacttc ccctgtgtta ctcaagaaac agctgtggca acgcacgctt cctggccccc 180 catcccttcc tccgtccctg ccctcccccg tctaccatct gctcagtgcc caggctggcc 240 acagccagtg cccagtgggt aaaacgctca aatgaggtag ccactgaatg gggcccttgg 300 tggccgggtg gggtggctgg ggtgggtggc cantgcancc acaggccctc acatacggnc 360 ctgtctgtgt gtcccgtgga acgctacntg gatgg 395 <210> SEQ ID NO 41 <211> LENGTH: 652 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 510, 533, 560, 623, 648 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 41 gaagagaggc atcctcaccc tgaagtaccc catcgagcac ggcatcgtca ccaactggga 60 cgacatggag aaaatctggc accacacctt ctacaatgag ctgcgtgtgg ctcccgagga 120 gcaccccgtg ctgctgaccg aggcccccct gaaccccaag gccaaccgcg agaagatgac 180 ccagatcatg tttgagacct tcaacacccc agccatgtac gttgctatcc aggctgtgct 240 atccctgtac gcctctggcc gtaccactgg catcgtgatg gactccggtg acggggtcac 300 ccacactgtg cccatctacg aggggtatgc cctcccccat gccatcctgc gtctggacct 360 ggctggccgg gacctgactg actacctcat gaagatcctc accgagcgcg gctacagctt 420 caccaccacg gccgagcggg aaatcgtgcg tgacattaag gagaagctgt gctacgtcgc 480 cctggacttc gagcaagaga tggccacggn ttgcttccag ctcctccctg ganaaagaac 540 tacgagcttg ccttgacggn caggtcatca ccattgggca aatgagccgg gttccgctgg 600 cccttgaagg cacttctttc canccctttc cttttcctgg gggcaatngg ag 652 <210> SEQ ID NO 42 <211> LENGTH: 413 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 42 nagggcgatg ggacagtgca ccgcaggaag aagaggagga cgtgcagcat ggtgggaaac 60 ggggacacca cctcccagga cgactgtgtc agcaaagagc gcagctcctc caggtgaccc 120 agcaaggctg ttgtctgtat ggaaggacac gctcgcggca agggcagggc ctggggaggg 180 tggcctgtcc agtcctgcag acaaggggag gcctgacaga gcccaagaat gaggacaccc 240 tcggcacggg aacccattca cttagcgttt gctccagtag ctttccctct gctaccaatg 300 cagataaacg cggcttgttt tactcaggca agagaatgtg aatagtgcca agaaaatcct 360 ttacattatt taataaaaat tgaatccatt ttcaaaaaaa aaaaaaaaaa aaa 413 <210> SEQ ID NO 43 <211> LENGTH: 622 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 547, 552, 610, 621 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 43 gcccaacaca atggcacgcc tgaaaagtgg gtgaggttca agtacccaaa gctcatctcc 60 aactacctga agaaagtcat ctcctactgg cggaatgaat ttgactggaa gaagcaggtg 120 gagattctca acagacaccc tcacttcaag actaagattg aagggctgga catccacttc 180 atccacgtga agccccccca gctgcccgca ggccataccc cgaagccctt gctgatggtg 240 cacggctggc ccggctcttt ctacgagttt tataagatca tcccactcct gactgacccc 300 aagaaccatg gcctgagcga tgagcacgtt tttgaagtca tctgcccttc catccctggc 360 tatggcttct cagaggcatc ctccaagaag gggttcaact cggtggccac cgccaggatc 420 ttttacaagc tgatgctgcg gctgggcttc caggcttttc ttggggaaga tccccttttc 480 tgaggaatga gtttgcctcc gtcccctgcc catgctggga agcccaccgc tcaccccctc 540 acccctncaa gntcactccc caccccccaa ctccgtgtgg gtaagcaaca tggcttttga 600 tgataaacgn acttttactt nt 622 <210> SEQ ID NO 44 <211> LENGTH: 614 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 518, 540, 592, 601 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 44 gaaagcccgc ctctgtccta aaatacaaac aagcacagac attaaacctg gatactatat 60 gataaagagg gatgtaacta ttgaattgga tacaaggatc agaatggaaa gaaactcacg 120 atgaaattga acctggtttt tgtatattta tcaaacttgt gctgagaata gtgtctgatt 180 atacgacttt taagcaaagt tgggtgtaat taggtgaaaa cagcccaggt cctcccggga 240 gcacagaggg gctaggggct ggtccttctc gtttgctcta gtcttgcttt gctgtctggt 300 gtagctcctc tgctgctccc atctgcacta attgacccaa aacgtgggta tttcctgcta 360 cacaaaagcc aaaaggtttc atgtagattt tagttcacta aagggtgccc acaaaataga 420 gattaatttt aacttaaatt ttaagcttga agattaggta ctatctgtga agttacactt 480 tttttttttt ttttttaaag gtagagatgt gtgtgtgngt aggtattaaa gatgtgttgn 540 tggtttccaa aaaggacact ggaaaataaa ttttgaatgg ttatgttctc anaatcaggg 600 ntgccagtcc cttg 614 <210> SEQ ID NO 45 <211> LENGTH: 390 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 gcccctcctt gggagaatcc cctagatcac agctcctcac catggactgg acctggagca 60 tccttttctt ggtggcagca gcaacaggtg tccactccca ggttcagctg gtgcagtctg 120 gacctgaggt gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct tccggttaca 180 catttaacag atatggtatc agctgggtgc gacaggcccc tggacaaggg cttgagtgga 240 tggggtggat cagtgcttac aacgggaaca cactctatgc acagaaattc cagggcagag 300 tcaccatgac cacagacaca cccacgagca cagcctacat ggaggtgagg agtctgagat 360 ctgacgactc ggccgtgtat tattgcgcga 390 <210> SEQ ID NO 46 <211> LENGTH: 686 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 601, 624, 650, 673 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 46 agtggcgaat ctcatctttg tctaacttcc cgtccttgtt cagatcccgg aattcgttaa 60 actgctcccg ttctgataaa acccagtctg gctcagggcc attctcctca tgggaaaaca 120 tatccgcaat atactcatcc tgatccacaa acccatcccc gttcttgtcg atgtcctcca 180 gggtttccaa aaccacaatt tccttcatat gttcaaactc ttcaggatgc agaaaggcag 240 tgaactcctc ccgagtagct gtcaggtcac cattgaggtc tgcagctttg aatcttctct 300 catcacgtgg cagcatcttt ttaaaggtgt gatgatctga agaatcatga aactctgcgg 360 ggtttcctag gtagtaacca taggtggctt gtttgtattc ttcccaggaa attttatcat 420 ccttgtccct atcataatcc ttccagactt tggcgacatt atcaaagatg tatcttttct 480 gcacccgttt gatccaggtt ttcagctcct cagtagtgac aaagccatcc ccatcattgt 540 cgattcgatc aacaatcttc ctagcctctc cttgtctcgt ccggggtgag ctgggcgaag 600 ncttggagtc ttcttgccaa gaanggctcg tggcgactgg aagttggtgn ctagggggcg 660 tcgccactcg atnggcgacc cctttg 686 <210> SEQ ID NO 47 <211> LENGTH: 561 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 544, 550 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 47 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 gganagcgan gattgcctgg a 561 <210> SEQ ID NO 48 <211> LENGTH: 411 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 33, 63, 280, 331, 350, 373, 391 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 48 gttctctgcg atgacataat atgtgacgat cangaattag actgccccaa cccagaaatt 60 ccntttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctn cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc ncaggctatc ctggaccagn tggcccccca 360 ggccctcccg ggncccctgg tacatctggt natcctggtt cccctggatc t 411 <210> SEQ ID NO 49 <211> LENGTH: 549 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 480, 498, 523, 539 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 49 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggn 480 gctatagggc catctggncc tgctggaaaa agaagggaga atnaggtaga ccccggacna 540 actgggaaa 549 <210> SEQ ID NO 50 <211> LENGTH: 547 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 438, 445, 515, 542 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 50 cattcgcagc ctttagcatc atgtagaagc aaactgcacc tatggctgag ataggtgcaa 60 tgacctacaa gattttgtgt tttctagctg tccaggaaaa gccatcttca gtcttgctga 120 cagtcaaaga gcaagtgaaa ccatttccag cctaaactac ataaaagcag ccgaaccaat 180 gattaaagac ctctaaggct ccataatcat cattaaatat gcccaaactc attgtgactt 240 tttattttat atacaggatt aaaatcaaca ttaaatcatc ttatttacat ggccatcggt 300 gctgaaattg agcattttaa atagtacagt aggctggtat acattaggaa atggactgca 360 ctggaggcaa atagaaaact aaagaaatta gataggctgg aaatgcttac tttctgctct 420 cattttcttt cattttgnct gctgnctttt tgaatgccaa tactttcatt taaaatggtt 480 ttgggagcac ggcaacccaa aatgactgaa cgggngggcc aaaaatccaa ccaactgata 540 antactt 547 <210> SEQ ID NO 51 <211> LENGTH: 617 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 581, 605 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 51 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcagg attgcctgga cctccaggta tcaaaggtcc ngctgggata cctggattcc 600 tggtntgaaa ggaccca 617 <210> SEQ ID NO 52 <211> LENGTH: 659 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 627 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 52 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcgag gattgcctgg acctccaggt atcaaaggtc cagctgggat acctggattc 600 cctggtatga aaggacacag aggcttngat gggacgaaat ggagaaaagg gtgaaacag 659 <210> SEQ ID NO 53 <211> LENGTH: 653 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 517, 579, 581, 603, 649 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 53 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatgganaat caggtagacc cggacgacct 540 ggaaaagcga ggatggctgg acctccaggt atcaaaggnc ngctgggata cctggattcc 600 tgnatgaaag gacacaaagg cttcatggac caaatgggaa aaaaggggna act 653 <210> SEQ ID NO 54 <211> LENGTH: 549 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 356, 363, 417, 433, 442, 461, 463, 464, 469, 479, 485, 489, 537, 545 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 54 cgacgaggtg gcccggcgct ggggagagcg gaagagcaaa cccaacatga actacgataa 60 gctcagccgc gccctccgtt actactatga caagaacatc atgaccaagg tccatgggaa 120 gcgctacgcc tacaagttcg acttccacgg gatcgcccag gccctccagc cccacccccc 180 ggagtcatct ctgtacaagt acccctcaga cctcccgtac atgggctcct atcacgccca 240 cccacagaag atgaactttg tggcgcccca ccctccagcc ctccccgtga catcttccag 300 tttttttgct gccccaaacc catactggaa ttcaccaact gggggtatat accccntcac 360 tangctcccc accagccata tgccttttca tctggggcac ttactactaa agacctngcg 420 gaggcttttc ccntcagcgt gnattcccag cccatcggcc ncnnaactnt tattcggana 480 acatnaatna aaaattgcct tcaagaggga ttgaaaaaaa gcttttactt ggggctnggg 540 gaaangaaa 549 <210> SEQ ID NO 55 <211> LENGTH: 207 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 55 caccaacaca gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg 60 tttgagagaa cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt 120 ccagaatgga agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc 180 aaagtgccgt agcttgtgta acccact 207 <210> SEQ ID NO 56 <211> LENGTH: 572 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 2, 467, 514, 541, 547 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 56 cntctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccag 360 gccctcccgg tccccctggt acatctggtc atcctggttc ccctggatct ccaggatacc 420 aaggaccccc tggtgaacct gggcaagctg gtcttcaggc cctccangac ctcctggtgc 480 tataggtcca tctggcctgc tggaaaaaaa gganaatcag gtagacccgg acgacctgga 540 nagcgangga ttgcctggac cctccaggta tt 572 <210> SEQ ID NO 57 <211> LENGTH: 425 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 27, 84, 328, 364, 389, 413 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 57 gaagcagccc atgctcaaat gaggcanaaa cctttgcttt taacacatag tatagctttg 60 taatcctttt ctgcacactc gggnaatcct tctttttcat tcctgatttt catgatatga 120 gtcttctttt ttcccctctg tcagtctagc taatggtttg tcaattttgt tgatcttttg 180 aagaacaaac ctttggttcc actttcttgt tgcatatgct gagtattctc ataattggag 240 tggaaagctg atctttgatt acttatttta cttacggctg aggagttcat ggacttcgca 300 aaacctcctt gaatctaaat tgcatctnct ttcctgggtc tgggctgata catgtatctt 360 cccncttata tacccttggg cttttcatnc gcggattaaa cactagagaa agncacaccc 420 ctgcc 425 <210> SEQ ID NO 58 <211> LENGTH: 325 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 323 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 58 ccgagcagtg ctaatgctgg ctactgtgca agcctgactt catgctcagg attcaagaaa 60 tggaaggatg aatcttgtga gaagaagttc tcctttgttt gcaagttcaa aaactagagg 120 aagctgaaaa atggatgtct agaactggtc ctgcaattac tatgaagtca aaaattaaac 180 tagactatgt ctccaactca gttcagacca tctcctccct aatgagtttg catcgctgat 240 cttcagtacc ttcacctgtc tcagtctcta gagccctgaa aaataaaaac aaacttattt 300 ttaaaaaaaa aaaaaaaaaa aanaa 325 <210> SEQ ID NO 59 <211> LENGTH: 207 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 59 caccaacaca gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg 60 tttgagagaa cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt 120 ccagaatgga agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc 180 aaagtgccgt agcttgtgta acccact 207 <210> SEQ ID NO 60 <211> LENGTH: 408 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 185, 293, 340, 371, 386 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 60 gctaaaaata caaaaaatta gctggctgtg gtggcgggcg cctgtaatcc cagctactcg 60 gaggcacacg caggagaatt gcttgaactc gggaggcaga ggttgcagtg agctgagatc 120 atgccactgc actccagcct gggcgacaag agcaaaactc cgtctcaaaa aagataaaga 180 aaaangaaaa atgtcacttt taaaaaaatt ggagtcctgt tcctccactt gccctctgga 240 tcttacccct tctcaccttt tcagaatctt cattcttccc tcttgcctgc agncatccca 300 tgtctcttgc atcttcagtt tctctattag atcattcctn tcacatgtaa atataacaga 360 attatcccct nttttattat tcattnctct aatcctctta acagcaaa 408 <210> SEQ ID NO 61 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 61 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 62 <211> LENGTH: 587 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 349, 456, 461, 513, 524, 580, 587 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 62 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccant ggccccccag 360 gccctcccgg tcccctggta catcttggtc atcctgggtt cccttggatc tccaggatac 420 caaggacccc ctggtgaacc ttgggcaaag ctggtncttt nggccctcca ggacctcctg 480 gtgctatagg gtccatcttg gtcctgctgg aanagatgga gaancaggta gacccggacg 540 acctggagag cgggggattg ccctgggcct ccagggttan aaggccn 587 <210> SEQ ID NO 63 <211> LENGTH: 193 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 63 gaaattaacc ccctaataaa attaattaac cactcactca tcgacctccc caccccatcc 60 aacatctccg catgatgaaa cttcggctca ctccttggcg cctgcctgat cctccaaatc 120 accacaggac tattcctagc catgcactac tcaccagacg cctcaaccgc cttttcatca 180 atcgcccaca tca 193 <210> SEQ ID NO 64 <211> LENGTH: 547 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 405 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 64 cattcgcagc ctttagcatc atgtagaagc aaactgcacc tatggctgag ataggtgcaa 60 tgacctacaa gattttgtgt tttctagctg tccaggaaaa gccatcttca gtcttgctga 120 cagtcaaaga gcaagtgaaa ccatttccag cctaaactac ataaaagcag ccgaaccaat 180 gattaaagac ctctaaggct ccataatcat cattaaatat gcccaaactc attgtgactt 240 tttattttat atacaggatt aaaatcaaca ttaaatcatc ttatttacat ggccatcggt 300 gctgaaattg agcattttaa atagtacagt aggctggtat acattaggaa atggactgca 360 ctggaggcaa atagaaacta aagaaattag atagggctgg gaaancttac tttctggctc 420 tcattttctt tcattttgtc tgctgtcttt ttgaatgcca atactttcat ttaaaatgtt 480 ttgggagcac ggcaacccaa aatgactgaa cggtggtcca aaaatccaaa ccaactgata 540 attactt 547 <210> SEQ ID NO 65 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 65 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 66 <211> LENGTH: 648 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 642, 646 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 66 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcgag gattgcctgg acctccaggt atcaaaggtc cagctgggat acctggattc 600 ctggtatgaa aggacacaga ggcttcgatg gacgaaatgg anaaangg 648 <210> SEQ ID NO 67 <211> LENGTH: 125 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 99, 101 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 67 gtaacttcgg gataaggatt ggctctaagg gctgggtcgg tcgggctggg gcgcgaagcg 60 gggctgggcg cgcgccgcgg ctggacgagg cgccgccgnc ncctcctatt ccggctacta 120 atggt 125 <210> SEQ ID NO 68 <211> LENGTH: 655 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 654 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 68 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcgag gattgcctgg acctccaggt atcaaaggtc cagctgggat acctggattc 600 cctggtatga aaggacacag agcttcgatg gacgaaatgg agaaaagggt gaant 655 <210> SEQ ID NO 69 <211> LENGTH: 656 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 565, 619, 621 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 69 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcgag gattgcctgg acctncaggt atcaaaggtc cacttgggat acctggattc 600 ctggtatgaa aggacacana ngcttcgatg gacgaaatgg aaaaaagggt gaaact 656 <210> SEQ ID NO 70 <211> LENGTH: 207 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 70 caccaacaca gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg 60 tttgagagaa cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt 120 ccagaatgga agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc 180 aaagtgccgt agcttgtgta acccact 207 <210> SEQ ID NO 71 <211> LENGTH: 658 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 71 gttctctgcg atgacataat atgtgacgat caagaattag actgccccaa cccagaaatt 60 ccatttggag aatgttgtgc agtttgccca cagcctccaa ctgctcctac tcgccctcct 120 aatggtcaag gacctcaagg ccccaaggga gatccaggcc ctcctggtat tcctgggaga 180 aatggtgacc ctggtattcc aggacaacca gggtcccctg gttctcctgg cccccctgga 240 atctgtgaat catgccctac tggtcctcag aactattctc cccagtatga ttcatatgat 300 gtcaagtctg gagtagcagt aggaggactc gcaggctatc ctggaccagc tggcccccca 360 ggccctcccg gtccccctgg tacatctggt catcctggtt cccctggatc tccaggatac 420 caaggacccc ctggtgaacc tgggcaagct ggtccttcag gccctccagg acctcctggt 480 gctataggtc catctggtcc tgctggaaaa gatggagaat caggtagacc cggacgacct 540 ggagagcgag gattgcctgg acctccaggt atcaaaggtc cagctgggat acctggattc 600 cctggtatga aaggacacag aggcttcgat ggacgaaatg garaaaaggg tgaaactg 658 <210> SEQ ID NO 72 <211> LENGTH: 550 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 517 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 72 cattcgcagc ctttagcatc atgtagaagc aaactgcacc tatggctgag ataggtgcaa 60 tgacctacaa gattttgtgt tttctagctg tccaggaaaa gccatcttca gtcttgctga 120 cagtcaaaga gcaagtgaaa ccatttccag cctaaactac ataaaagcag ccgaaccaat 180 gattaaagac ctctaaggct ccataatcat cattaaatat gcccaaactc attgtgactt 240 tttattttat atacaggatt aaaatcaaca ttaaatcatc ttatttacat ggccatcggt 300 gctgaaattg agcattttaa atagtacagt aggctggtat acattaggaa atggactgca 360 ctggaggcaa atagaaaact aaagaaatta gatagggctg graawgctta ctttctggct 420 ctcattttct ttcattttgt ctgctgtctt tttgaatgcc aatactttca tttaaaatgg 480 ttttgggagc acggcaaccc aaaatgactg aacggknggk ccaaaaatcc aaaccaactg 540 ataattactt 550 <210> SEQ ID NO 73 <211> LENGTH: 766 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 44, 48, 737, 764 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 73 ggacggaact gccacgatgc tgccactttg gactctttca ctgntggngg gagcagtagc 60 aggaaaagaa gtttgctacg aaagactcgg ctgcttcagt gatgactccc catggtcagg 120 aattacggaa agacccctcc atatattgcc ttggtctcca aaagatgtca acacccgctt 180 cctcctatat actaatgaga acccaaacaa ctttcaagaa gttgccgcag attcatcaag 240 catcagtggc tccaatttca aaacaaatag aaaaactcgc tttattattc atggattcat 300 agacaaggga gaagaaaact ggctggccaa tgtgtgcaag aatctgttca aggtggaaag 360 tgtgaactgt atctgtgtgg actggaaagg tggctcccga actggataca cacaagcctc 420 gcagaacatc aggatcgtgg gagcagaagt ggcatatttt gttgaatttc ttcagtcggc 480 gttcggttac tcaccttcca acgtgcatgt cattggccac agcctgggtg cccacgctgc 540 tggggaggct ggaaggagaa ccaatgggac cattggacgc atcacagggt tggacccagc 600 agaaccttgc tttcagggca cacctgaatt agtccgattg gaccccaccg atgccaaatt 660 tgtggatgta attcacacgg atggtgcccc catagtcccc caatttgggg tttgggaatg 720 agccaagtcg tgggccncct tgaattcttt ccaaatggga gagngg 766 <210> SEQ ID NO 74 <211> LENGTH: 761 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 3, 10, 93, 106, 107, 652, 687, 706, 759, 761 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 74 ccnttcccan attccgtcta tgtccacaat ctgagagaga atgttctttt tacatccagg 60 catttccact cctccatttg gaaagaaatc tangtggccc acgggnnggg tcattccaaa 120 ccccaaattg gggactatgg gggcaccatc cgtgtgaatt acatccacaa atttggcatc 180 gctggggtcc aatcggacta attcaggtgt gccctgaaag caaggttctg ctgggtccaa 240 ccctgtgatg cgtccaatgg tcccattggt tctccttcca gcctccccag cagcgtgggc 300 acccaggctg tggccaatga catgcacgtt ggaaggtgag taaccgaacg ccgactgaag 360 aaattcaaca aaatatgcca cttctgctcc cacgatcctg atgttctgcg aggcttgtgt 420 gtatccagtt cgggagccac ctttccagtc cacacagata cagttcacac tttccacctt 480 gaacagattc ttgcacacat tggccagcca gttttcttct cccttgtcta tgaatccatg 540 aataataaag cgagtttttc tatttgtttt gaaattggag ccactgatgc ttgatgaatc 600 tgcggcaact tcttgaaagt tgtttgggtt ctcattagta tataggagga ancggggtgt 660 tggacatctt tttggagacc aaggcantat atgggagggg tctttnccgt aattccctga 720 ccatggggga gtcatcactg gaaccggccg agccttttnt n 761 <210> SEQ ID NO 75 <211> LENGTH: 789 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 54, 96, 97, 227, 244, 332, 358, 373, 435, 552, 571, 573, 587, 613, 680, 691, 726, 739, 774, 775 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 75 cggaactgcc acgatgctgc cactttggac tctttcactg ctgctgggag cagnagcagg 60 aaaagaagtt tgctacgaaa gactcggctg cttcanngat gactccccat ggtcaggaat 120 tacggaaaga cccctccata tattgccttg gtctccaaaa gatgtcaaca cccgcttcct 180 cctatatact aatgagaacc caaacaactt tcaagaagtt gccgcanatt catcaagcat 240 cagnggctcc aatttcaaaa caaatagaaa aactcgcttt attattcatg gattcataga 300 caagggagaa gaaaactggc tggccaatgt gngcaagaat ctgttcaagg tggaaagngt 360 gaactgtatc tgngtggact ggaaaggtgg ctcccgaact ggatacacac aagcctcgca 420 gaacatcagg atcgngggag cagaagtggc atattttgtt gaatttcttc agtcggcgtt 480 cggttactca ccttccaacg tgcatgtcat tggccacagc ctgggtgccc acgctgctgg 540 ggaggctgga angagaacca atgggaccat ngnacgcatc acagggntgg acccagcaga 600 accttgcttt canggcacac ctgaattagt cgattggacc ccacgatgcc aaatttgtgg 660 atgtaattca cacggatggn gcccccatat ncccaatttt ggggtttgga atgaacccaa 720 gtccgngggg ccaccttana tttctttttc caaaagggag ggaaatggga aaannggccc 780 tggaatggt 789 <210> SEQ ID NO 76 <211> LENGTH: 907 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 23, 104, 110, 763, 786, 792, 819, 822, 834, 836, 842, 843, 892, 900 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 76 ctcttgccag attccgtcta tgnccacatt ctgatagaga atgttctttt tacatccagg 60 catttccact cctccatttg gaaagaaatc taggtggccc acgncttggn tcattccaaa 120 ccccaaattg gggactatgg gggcaccatc cgtgtgaatt acatccacaa atttggcatc 180 gctggggtcc aatcggacta attcaggtgt gccctgaaag caaggttctg ctgggtccaa 240 ccctgtgatg cgtccaatgg tcccattggt tctccttcca gcctccccag cagcgtgggc 300 acccaggctg tggccaatga catgcacgtt ggaaggtgag taaccgaacg ccgactgaag 360 aaattcaaca aaatatgcca cttctgctcc cacgatcctg atgttctgcg aggcttgtgt 420 gtatccagtt cgggagccac ctttccagtc cacacagata cagttcacac tttccacctt 480 gaacagattc ttgcacacat tggccagcca gttttcttct cccttgtcta tgaatccatg 540 aataataaag cgagtttttc tatttgtttt gaaattggag ccactgatgc ttgatgaatc 600 tgcggcaact tcttgaaagt tgtttgggtt ctcattagta tataggagga agcgggtgtt 660 gacatctttt ggagaccaag gcaatatatg gaggggtctt tcccgtaatt ctgaccatgg 720 ggagtcatca ctgaagcagc cgagtctttc gtacaaaact ttntttttcc tgctactggt 780 tcccangaac anggggaaag gagtcccaaa agtgggcang cnattcgtgg ggcnantttc 840 cnnccaacca caattgggaa attccccccc cccttgggac ttagggggga anccccctan 900 tttctta 907 <210> SEQ ID NO 77 <211> LENGTH: 750 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 45, 46, 53, 685, 722, 730 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 77 gcggaactgc cacgatgctg ccactttgga ctctttcact gctgnnggga gcngtagcag 60 gaaaagaagt ttgctacgaa agactcggct gcttcagtga tgactcccca tggtcaggaa 120 ttacggaaag acccctccat atattgcctt ggtctccaaa agatgtcaac acccgcttcc 180 tcctatatac taatgagaac ccaaacaact ttcaagaagt tgccgcagat tcatcaagca 240 tcagtggctc caatttcaaa acaaatagaa aaactcgctt tattattcat ggattcatag 300 acaagggaga agaaaactgg ctggccaatg tgtgcaagaa tctgttcaag gtggaaagtg 360 tgaactgtat ctgtgtggac tggaaaggtg gctcccgaac tggatacaca caagcctcgc 420 agaacatcag gatcgtggga gcagaagtgg catattttgt tgaatttctt cagtcggcgt 480 tcggttactc accttccaac gtgcatgtca ttggccacag cctgggtgcc cacgctgctg 540 gggaggctgg aaggagaacc aatgggacca ttggacgcat cacagggttg gacccagcag 600 aaccttgctt tcagggcaca cctgaattag tccgattgga ccccaacgat gccaaatttt 660 gtggatgtaa ttccacggat ggggncccca tagtccccca tttggggttg gaatgagcca 720 antcgtgggn cacctagatt tctttccaaa 750 <210> SEQ ID NO 78 <211> LENGTH: 764 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1, 94, 102, 107, 108, 662, 712, 745, 760 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 78 ncccttccca gattccgtct atgtccacaa tctgagagag aatgttcttt ttacatccag 60 gcatttccac tcctccattt ggaaagaaat ctangtggcc cncgggnngg gtcattccaa 120 accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca aatttggcat 180 cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct gctgggtcca 240 accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca gcagcgtggg 300 cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac gccgactgaa 360 gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc gaggcttgtg 420 tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca ctttccacct 480 tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct atgaatccat 540 gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg cttgatgaat 600 ctggcggcaa cttcttgaaa gttgtttggg ttctcattag tatataggag gaagcgggtg 660 tngacatctt ttggagacca aggcaatata tgggaggggt ctttccgtaa tncctgacca 720 tgggggagtc atcactggaa gcagncagtt ttttcctaan aaaa 764 <210> SEQ ID NO 79 <211> LENGTH: 651 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 45, 46, 52, 414, 554, 590, 617, 639 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 79 gcggaactgc cacgatgctg ccactttgga ctctttcact gctgnnggga gnagtagcag 60 gaaaagaagt ttgctacgaa agactcggct gcttcagtga tgactcccca tggtcaggaa 120 ttacggaaag acccctccat atattgcctt ggtctccaaa agatgtcaac acccgcttcc 180 tcctatatac taatgagaac ccaaacaact ttcaagaagt tgccgcagat tcatcaagca 240 tcagtggctc caatttcaaa acaaatagaa aaactcgctt tattattcat ggattcatag 300 acaagggaga agaaaactgg ctggccaatg tgtgcaagaa tctgttcaag gtggaaagtg 360 tgaactgtat ctgtgtggac tggaaaggtg gctcccgaac tggatacaca caancctcgc 420 agaacatcag gatcgtggga gcagaagtgg catattttgt tgaatttctt cagtcggcgt 480 tcggttactc accttccaac gtgcatgtca ttggccacag cctgggtgcc cacgctgctg 540 ggggaggctg gaangagaac caatgggacc attgggacgc atcacagggn tggacccacc 600 agaacctttg ctttcanggg ccaccttgaa ttagttccna ttgggacccc a 651 <210> SEQ ID NO 80 <211> LENGTH: 741 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1, 13, 83, 84, 86, 92, 602, 630, 718, 726 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 80 natgtccaca atntgagaga gaatgttctt tttacatcca ggcatttcca ctcctccatt 60 tggaaagaaa tctaggtggc ccnngngttg gntcattcca aaccccaaat tggggactat 120 gggggcacca tccgtgtgaa ttacatccac aaatttggca tcgctggggt ccaatcggac 180 taattcaggt gtgccctgaa agcaaggttc tgctgggtcc aaccctgtga tgcgtccaat 240 ggtcccattg gttctccttc cagcctcccc agcagcgtgg gcacccaggc tgtggccaat 300 gacatgcacg ttggaaggtg agtaaccgaa cgccgactga agaaattcaa caaaatatgc 360 cacttctgct cccacgatcc tgatgttctg cgaggcttgt gtgtatccag ttcgggagcc 420 acctttccag tccacacaga tacagttcac actttccacc ttgaacagat tcttgcacac 480 attggccagc cagttttctt ctcccttgtc tatgaatcca tgaataataa agcgagtttt 540 tctatttgtt ttgaaattgg agccactgat gcttgatgaa tctgcggcaa cttcttgaaa 600 gntggttggg ttctcattag tatataggan gaagccgggg gttgacatct tttggagacc 660 aaggcaatat atggaggggg tcttttccgt aattcctgac catgggggag tcatcacntg 720 aagcangccc gaattctttt c 741 <210> SEQ ID NO 81 <211> LENGTH: 967 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 48, 498, 572, 598, 619, 634, 641, 654, 659, 661, 670, 675, 713, 714, 726, 735, 743, 751, 756, 772, 793, 795, 804, 805, 806, 808, 809, 811, 840, 855, 856, 869, 881, 884, 892, 894, 895, 898, 899, 912, 948, 958 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 81 ggacggaact gccacgatgc tgccactttg gactctttca ctgctggngg gagcagtagc 60 aggaaaagaa gtttgctacg aaagactcgg ctgcttcagt gatgactccc catggtcagg 120 aattacggaa agacccctcc atatattgcc ttggtctcca aaagatgtca acacccgctt 180 cctcctatat actaatgaga acccaaacaa ctttcaagaa gttgccgcag attcatcaag 240 catcagtggc tccaatttca aaacaaatag aaaaactcgc tttattattc atggattcat 300 agacaaggga gaagaaaact ggctggccaa tgtgtgcaag aatctgttca aggtggaaag 360 tgtgaactgt atctgtgtgg actggaaagg tggctcccga actggataca cacaagcctc 420 gcagaacatc aggatcgtgg gagcagaagt ggcatatttt gttgaatttc ttcagtcggc 480 gttcggttac tcaccttnca accgtgcatg tcattggcca cagcctgggt gcccaccctt 540 gcttggggga agctggaaag gagaacccaa tngggaccat tgggaccgcc ttcaccangg 600 gttgggaccc agcaaaaanc ctttgctttt tcanggggca ncacccctgg aaanttaant 660 ncccgaattn ggganccccc agcggaatgg cccaaaattt tgggggggaa tgnnaaaatt 720 ccaccnccgg ggatnggggg ggnccccccc ntaaantccc cccaaatttt tngggggggt 780 tttgggaaaa agnanacccc aaannntnng nggggggccc cccccccaaa aaaatttttn 840 ttttttcccc aaaanngggg gggggaaang gggggaaaaa nggnccccgg gnannggnna 900 aaaaaaaaaa anaatttttt ttttttttca aaaaaggggg ggaccaanaa aaaggaanat 960 tttgggg 967 <210> SEQ ID NO 82 <211> LENGTH: 735 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 2, 10, 52, 54, 60, 131, 633, 644, 703, 726 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 82 tncatccaan catttccact cctccatttg gaaagaaatc tatgtggccc angngttggn 60 tcattccaaa ccccaaattg gggactatgg gggcaccatc cgtgtgaatt acatccacaa 120 atttggcatc nctggggtcc aatcggacta attcaggtgt gccctgaaag caaggttctg 180 ctgggtccaa ccctgtgatg cgtccaatgg tcccattggt tctccttcca gcctccccag 240 cagcgtgggc acccaggctg tggccaatga catgcacgtt ggaaggtgag taaccgaacg 300 ccgactgaag aaattcaaca aaatatgcca cttctgctcc cacgatcctg atgttctgcg 360 aggcttgtgt gtatccagtt cgggagccac ctttccagtc cacacagata cagttcacac 420 tttccacctt gaacagattc ttgcacacat tggccagcca gttttcttct cccttgtcta 480 tgaatccatg aataataaag cgagtttttc tatttgtttt gaaattggag ccactgatgc 540 ttgatgaatc tgcggcaact tcttgaaagt tgtttgggtt ctcattagta tataggagga 600 agcgggtggt tgacatcttt ttgagaccaa ggnaatatat ggangggtct tttccgtatt 660 cctggacctg ggggagtcat cactgaagca gccgaatctt tcntagcaac ttcttttctg 720 ctctgnttcc acaag 735 <210> SEQ ID NO 83 <211> LENGTH: 560 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 2, 29, 99, 105 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 83 cncacagaac atgttgttgg taatctttnc agggtaggag gcttcacact cagcctggct 60 cagcacagga gcatccaggc actgcagctc gtctgggtng tcggnaccag aactcagagt 120 gttgccccag ccggagatga gggactcggt gccagcagct ggaggggcag tgggcagaga 180 gatggcggac acgcgggaat tgatgacggc aggtgaggag agcttgatca gcaggatgtc 240 attgtccaga gtccggctgt tgtatttggg gtggcggatg atcttggctg cattgatgaa 300 ctgttcattc ccctccagga cttcgatgtt gtgctctccc agtctcacct ggatgcggga 360 cttgtagcag tgacctgctg acaccaccca ctgttcgctg atgagggagc caccgcagaa 420 gtggtagcca gaattcaagg acacctggta ggggacagaa ttctcctcac agatgtagcc 480 cccaacgatc ttgtcatcat catcaaaggg ggcagcaaca gcagctgcaa caaaggtaag 540 gatcagaagt agattcatgc 560 <210> SEQ ID NO 84 <211> LENGTH: 569 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 37, 40, 51, 53, 55, 66, 67, 79, 84, 86, 95, 111, 114, 125, 150, 153, 156, 159, 164, 166, 186, 200, 216, 230, 241, 245, 255, 268, 282, 294, 302, 311, 343, 346, 349, 356, 358, 366, 371, 383, 407, 415, 425, 470, 473, 475, 484, 493, 498 <223> OTHER INFORMATION: n = A,T,C or G <221> NAME/KEY: misc_feature <222> LOCATION: 502, 511, 512, 559, 569 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 84 gaactgccac gatgctgcca ctttggactc tttcacngcn ggtgggagca ntngnaggaa 60 aagaannttg ctacgaaana ctcngntgct tcagngatga ctccccatgg ncangaatta 120 cgganagacc cctccatata ttgccttggn ctncanaana tgtnancacc cgcttcctcc 180 tatatnctaa tgagaacccn aacaactttc aagaanttgc cgcatattcn tcaagcatca 240 ntggntccaa tttcnaaaca aatagaanaa ctctctttat tnttcatgga ttcntagaca 300 anggagaaga naactggctg gccaatgtgt gcaagaatct gtncanggng gaaagngnga 360 actgtntctg ngtggactgg aanggtggct cccgaactgg atacacncaa gcctnacaga 420 acatnaggat cgtgggagca gaagtggcat attttgttga atttcttcan tcngngttcg 480 gttnctcacc ttncaacntg cntgtcattg nncacagcct gggtgcccac gctgctgggg 540 aggctggaag gagaaccant gggaccatn 569 <210> SEQ ID NO 85 <211> LENGTH: 780 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 7, 8, 91, 100, 698, 769, 775 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 85 attccgnnta tgtccacaat ctgagagaga atgttctttt tacatccagg catttccact 60 cctccatttg gaaagaaatc taggtggccc ncgagttggn tcattccaaa ccccaaattg 120 gggactatgg gggcaccatc cgtgtgaatt acatccacaa atttggcatc gctggggtcc 180 aatcggacta attcaggtgt gccctgaaag caaggttctg ctgggtccaa ccctgtgatg 240 cgtccaatgg tcccattggt tctccttcca gcctccccag cagcgtgggc acccaggctg 300 tggccaatga catgcacgtt ggaaggtgag taaccgaacg ccgactgaag aaattcaaca 360 aaatatgcca cttctgctcc cacgatcctg atgttctgcg aggcttgtgt gtatccagtt 420 cgggagccac ctttccagtc cacacagata cagttcacac tttccacctt gaacagattc 480 ttgcacacat tggccagcca gttttcttct cccttgtcta tgaatccatg aataataaag 540 cgagtttttc tatttgtttt gaaattggag ccactgatgc ttgatgaatc ttgcggcaac 600 ttcttgaaag ttgtttgggt tctcattagt atataggagg aagcgggtgt tgacatcttt 660 tgggagacca aggcaatata tggagggggt ctttccgnaa ttccttgacc atgggggagt 720 catcacttga agcaaccgaa gtctttcgta gcaaaacttc tttttctgnt actgnttcca 780 <210> SEQ ID NO 86 <211> LENGTH: 556 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 86 gaagccccac agaacatgtt gttggtaatc tttccagggt aggaggcttc acactcagcc 60 tggctcagca caggagcatc caggcactgc agctcgtctg ggtagtcggc accagaactc 120 agagtgttgc cccagccgga gatgagggac tcggtgccag cagctggagg ggcagtgggc 180 agagagatgg cggacacgcg ggaattgatg acggcaggtg aggagagctt gatcagcagg 240 atgtcattgt ccagagtccg gctgttgtat ttggggtggc ggatgatctt ggctgcattg 300 atgaactgtt cattcccctc caggacttcg atgttgtgct ctcccagtct cacctggatg 360 cgggacttgt agcagtgacc tgctgacacc acccactgtt cgctgatgag ggagccaccg 420 cagaagtggt agccagaatt caaggacacc tggtagggga cagaattctc ctcacagatg 480 tagcccccaa cgatcttgtc atcatcatca aagggggcag caacagcagc tgcaacaaag 540 gtaaggatca gaagtc 556 <210> SEQ ID NO 87 <211> LENGTH: 752 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 661, 685, 691, 740, 741 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 87 gacctgacgg aactgccacg atgctgccac tttggactct ttcactgctg ctgggagcag 60 tagcaggaaa agaagtttgc tacgaaagac tcggctgctt cagtgatgac tccccatggt 120 caggaattac ggaaagaccc ctccatatat tgccttggtc tccaaaagat gtcaacaccc 180 gcttcctcct atatactaat gagaacccaa acaactttca agaagttgcc gcagattcat 240 caagcatcag tggctccaat ttcaaaacaa atagaaaaac tcgctttatt attcatggat 300 tcatagacaa gggagaagaa aactggctgg ccaatgtgtg caagaatctg ttcaaggtgg 360 aaagtgtgaa ctgtatctgt gtggactgga aaggtggctc ccgaactgga tacacacaag 420 cctcgcagaa catcaggatc gtgggagcag aagtggcata ttttgttgaa tttcttcagt 480 cggcgttcgg ttactcacct tccaacgtgc atgtcattgg ccacagcctg ggtgcccacg 540 ctgctgggga ggctggaagg agaaccaatg ggaccattgg acgcatcaca gggttggacc 600 cagcagaacc ttgctttcag ggcacacctg aattaagtcc gattggaccc cagcgatgcc 660 naatttgtgg gatgtaattc acacngatgg nggcccccat agtcccccaa tttgggggtt 720 tgggaatgag cccaaatcgn ngggcccccc ta 752 <210> SEQ ID NO 88 <211> LENGTH: 804 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 24, 106, 650, 698, 717, 775, 788 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 88 tcccttccca gattccgtct atgnccacaa tctgagagag aatgttcttt ttacatccag 60 gcatttccac tcctccattt ggaaagaaat ctaggtggcc cacganttgg ctcattccaa 120 accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca aatttggcat 180 cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct gctgggtcca 240 accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca gcagcgtggg 300 cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac gccgactgaa 360 gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc gaggcttgtg 420 tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca ctttccacct 480 tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct atgaatccat 540 gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg cttgatgaat 600 ctgcggcaac ttcttgaaag ttggttgggt tctcattagt atattaggan gaaagcgggg 660 tggttgacat cttttggaga ccaaggcaat atatggangg ggtcttttcc gtaattnctg 720 accatggggg agtcttactg aacaggcgaa tctttcgtag caaacttttt tctgntctgt 780 tccacagnaa tgaaagatcc aaag 804 <210> SEQ ID NO 89 <211> LENGTH: 208 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 48, 49 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 89 gcagttccct cctccttgtg gccgttgcct caggctatgg cccacctnnc tctcactctt 60 ccagccgcgt tgtccatggt gaggatgcgg tcccctacag ctggccctgg caggtttccc 120 tgcagtatga gaaaagtgga agcttctacc acacgtgtgg cggtagcctc atcgcccccg 180 attgggttgt gactgccggc cactgcat 208 <210> SEQ ID NO 90 <211> LENGTH: 230 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 127 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 90 ttggtccggc cccccctcga gatgcagtgg ccggcagtca caacccaatc gggggcgatg 60 aggctaccgc cacacgtgtg gtatgaagct tccacttttc tcatactgca gggaaacctg 120 ccagggncag ctgtagggga ccgcatcctc accatggaca acgcggctgg aagagtgaga 180 ggaaggtggg ccatagcctg aggcaacggc cacaaggagg agggaactgc 230 <210> SEQ ID NO 91 <211> LENGTH: 522 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 3, 5 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 91 gcngncccct ttgatgatga tgacaagatc gttgggggct acatctgtga ggagaattct 60 gtcccctacc aggtgtcctt gaattctggc taccacttct gcggtggctc cctcatcagc 120 gaacagtggg tggtgtcagc aggtcactgc tacaagtccc gcatccaggt gagactggga 180 gagcacaaca tcgaagtcct ggaggggaat gaacagttca tcaatgcagc caagatcatc 240 cgccacccca aatacaacag ccggactctg gacaatgaca tcctgctgat caagctctcc 300 tcacctgccg tcatcaattc ccgcgtgtcc gccatctctc tgcccactgc ccctccagct 360 gctggcaccg agtccctcat ctccggctgg ggcaacactc tgagttctgg tgccgactac 420 ccagacgagc tgcagtgcct ggatgctcct gtgctgagcc aggctgagtg tgaagcctcc 480 taccctggaa agattaccaa caacatgttc tgtgtgggct tc 522 <210> SEQ ID NO 92 <211> LENGTH: 565 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 103, 106 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 92 gaagccccac agaacatgtt gttggtaatc tttccagggt aggaggcttc acactcagcc 60 tggctcagca caggagcatc caggcactgc agctcgtctg ggnggncggg accagaactc 120 agagtgttgc cccagccgga gatgagggac tcggtgccag cagctggagg ggcagtgggc 180 agagagatgg cggacacgcg ggaattgatg acggcaggtg aggagagctt gatcagcagg 240 atgtcattgt ccagagtccg gctgttgtat ttggggtggc ggatgatctt ggctgcattg 300 atgaactgtt cattcccctc caggacttcg atgttgtgct ctcccagtct cacctggatg 360 cgggacttgt agcagtgacc tgctgacacc acccactgtt cgctgatgag ggagccaccg 420 cagaagtggt agccagaatt caaggacacc tggtagggga cagaattctc ctcacagatg 480 tagcccccaa cgatcttgtc atcatcatca aagggggcag caacagcagc tgcaacaaag 540 gtaaggatca gaagtagatt catgc 565 <210> SEQ ID NO 93 <211> LENGTH: 783 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 48, 448, 449, 453, 455, 466, 499, 502, 509, 521, 541, 612, 615, 616, 633, 640, 651, 658, 660, 683, 689, 708, 714, 717, 720, 723, 751, 753 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 93 ggacggaact gccacgatgc tgccactttg gactctttca ctgctggngg gagcagtagc 60 aggaaaagaa gtttgctacg aaagactcgg ctgcttcagt gatgactccc catggtcagg 120 aattacggaa agacccctcc atatattgcc ttggtctcca aaagatgtca acacccgctt 180 cctcctatat actaatgaga acccaaacaa ctttcaagaa gttgccgcag attcatcaag 240 catcagtggc tccaatttca aaacaaatag aaaaactcgc tttattattc atggattcat 300 agacaaggga gaagaaaact ggctggccaa tgtgtgcaag aatctgttca aggtggaaag 360 tgtgaactgt atctgtgtgg actggaaagg tggctcccga actggataca cacaagcctc 420 gcagaacatc aggatcgtgg gagcaganng ggntnttttg gtgaanttct tcaatcgggg 480 gtcggggtac ttcctttcna cntgcatgnt atttggccac ngcctgggtg cccacccttt 540 ntggggaggc ttggaaagag aaaccaatgg ggaccccttg ggaccccatt accagggggt 600 tgggacccca ancannaacc ctttgctttt tcngggggcn ccccccctgg natttaantn 660 cccgaattgg ggacccccca ccngaatgnc ccaaaatttt gggggggntt gttnaanttn 720 cancaccggg atggggggcc cccccattaa ntncccccca aattttgggg ggtttgggga 780 aaa 783 <210> SEQ ID NO 94 <211> LENGTH: 879 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 106, 111, 649, 711, 779, 788, 808, 811, 812, 817, 820, 825, 845, 851 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 94 tcccttccca gattccgtct atgtccacaa tctgagagag aatgttcttt ttacatccag 60 gcatttccac tcctccattt ggaaagaaat ctaggtggcc cacganttgg ntcattccaa 120 accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca aatttggcat 180 cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct gctgggtcca 240 accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca gcagcgtggg 300 cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac gccgactgaa 360 gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc gaggcttgtg 420 tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca ctttccacct 480 tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct atgaatccat 540 gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg cttgatgaat 600 ctgcggcaac ttcttgaaag ttgtttgggt tctcattagt atataggang aagcgggtgt 660 tgacatcttt tggagaccaa ggcaatatat ggaggggtct ttccgtaatt nctgaccatg 720 gggagtcatc acttgaagca gccgagtctt ttcgtacaaa cttctttttc tgctactgnt 780 cccagcanga gtggaaagag tccaaagngg nngcatncgn ggcanttccg tccccaacca 840 atggnatttc ncccccctgg gctaggggat cccttattt 879 <210> SEQ ID NO 95 <211> LENGTH: 577 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 48, 50, 51, 314, 357, 394, 402, 448, 455, 494, 540, 565 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 95 gcctgacgga actgccacga tgctgccact ttggactctt tcactggngn ngggagcagt 60 agcaggaaaa gaagtttgct acgaaagact cggctgcttc agtgatgact ccccatggtc 120 aggaattacg gaaagacccc tccatatatt gccttggtct ccaaaagatg tcaacacccg 180 cttcctccta tatactaatg agaacccaaa caactttcaa gaagttgccg cagattcatc 240 aagcatcagt ggctccaatt tcaaaacaaa tagaaaaact cgctttatta ttcatggatt 300 catagacaag gganaaaaaa actggctggc caatgtgtgc aaaaatctgt tcaaggngga 360 aagtgtgaac tgtatctgtg tggactggaa aggnggctcc cnaactggat accacacaag 420 cctcgcagaa cattaaggat cgtggganca gaagnggcat attttgttga atttcttcag 480 tcggcgttcg ggtnctcacc tttcaacgtg catgtcattg gccacagcct gggtgccccn 540 ctgcttgggg aaggctggaa agganaacca tggggac 577 <210> SEQ ID NO 96 <211> LENGTH: 748 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1, 102, 108, 698, 737, 745 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 96 ncccttccca gattccgtct atgtccacaa tctgagagag aatgttcttt ttacatccag 60 gcatttccac tcctccattt ggaaagaaat ctaggtggcc cncgggtngg gtcattccaa 120 accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca aatttggcat 180 cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct gctgggtcca 240 accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca gcagcgtggg 300 cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac gccgactgaa 360 gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc gaggcttgtg 420 tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca ctttccacct 480 tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct atgaatccat 540 gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg cttgatgaat 600 ctgcgggcaa cttcttgaaa agttgtttgg gttctcatta gtatatagga ggaaagcggg 660 tgttggacat cttttggaga ccaagggaat atatggangg gggctttccg taattcctga 720 ccatggggga gttcatnact tgaancag 748 <210> SEQ ID NO 97 <211> LENGTH: 534 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 2, 8 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 97 gnggctgntg ttgctgcccc ctttgatgat gatgacaaga tcgttggggg ctacatctgt 60 gaggagaatt ctgtccccta ccaggtgtcc ttgaattctg gctaccactt ctgcggtggc 120 tccctcatca gcgaacagtg ggtggtgtca gcaggtcact gctacaagtc ccgcatccag 180 gtgagactgg gagagcacaa catcgaagtc ctggagggga atgaacagtt catcaatgca 240 gccaagatca tccgccaccc caaatacaac agccggactc tggacaatga catcctgctg 300 atcaagctct cctcacctgc cgtcatcaat tcccgcgtgt ccgccatctc tctgcccact 360 gcccctccag ctgctggcac cgagtccctc atctccggct ggggcaacac tctgagttct 420 ggtgccgact acccagacga gctgcagtgc ctggatgctc ctgtgctgag ccaggctgag 480 tgtgaagcct cctaccctgg aaagattacc aacaacatgt tctgtgtggg cttc 534 <210> SEQ ID NO 98 <211> LENGTH: 566 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 gaagcccaca cagaacatgt tgttggtaat ctttccaggg taggaggctt cacactcagc 60 ctggctcagc acaggagcat ccaggcactg cggctggtct gggtagtcgg caccagaact 120 cagagtgttg ccccagccgg agatgaggga ctcggtgcca gcagctggag gggcagtggg 180 cagagagatg gcggacacgc gggaattgat gacggcaggt gaggagagct tgatcagcag 240 gatgtcattg tccagagtcc ggctgttgta tttggggtgg cggatgatct tggctgcatt 300 gatgaactgt tcattcccct ccaggacttc gatgttgtgc tctcccagtc tcacctggat 360 gcgggacttg tagcagtgac ctgctgacac cacccactgt tcgctgatga gggagccacc 420 gcagaagtgg tagccagaat tcaaggacac ctggtagggg acagaattct cctcacagat 480 gtagccccca acgatcttgt catcatcatc aaagggggca gcaacagcag ctgcaacaaa 540 ggtaaggatc agaagtagat tcatgc 566 <210> SEQ ID NO 99 <211> LENGTH: 566 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 35, 40, 468 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 99 gcatgaatct acttctgatc cttacctttg ttgcngctgn tgttgctgcc ccctttgatg 60 atgatgacaa gatcgttggg ggctacatct gtgaggagaa ttctgtcccc taccaggtgt 120 ccttgaattc tggctaccac ttctgcggtg gctccctcat cagcgaacag tgggtggtgt 180 cagcaggtca ctgctacaag tcccgcatcc aggtgagact gggagagcac aacatcgaag 240 tcctggaggg gaatgaacag ttcatcaatg cagccaagat catccgccac cccaaataca 300 acagccggac tctggacaat gacatcctgc tgatcaagct ctcctcacct gccgtcatca 360 attcccgcgt gtccgccatc tctctgccca ctgcccctcc agctgctggc accgagtccc 420 tcatctccgg ctggggcaac actctgagtt ctggtgccga ctacccanac gagctgcagt 480 gcctggatgc tcctgtgctg agccaggctg agtgtgaagc ctcctaccct ggaaagatta 540 ccaacaacat gttctgtgtg ggcttc 566 <210> SEQ ID NO 100 <211> LENGTH: 566 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 94, 98, 454, 457, 478, 484 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 100 gaagcccaca cagaacatgt tgttggtaat ctttccaggg taggaggctt cacactcagc 60 ctggctcagc acaggagcat ccaggcactg cggntggnct gggtagtcgg caccagaact 120 cagagtgttg ccccagccgg agatgaggga ctcggtgcca gcagctggag gggcagtggg 180 cagagagatg gcggacacgc gggaattgat gacggcaggt gaggagagct tgatcagcag 240 gatgtcattg tccagagtcc ggctgttgta tttggggtgg cggatgatct tggctgcatt 300 gatgaactgt tcattcccct ccaggacttc gatgttgtgc tctcccagtc tcacctggat 360 gcgggacttg tagcagtgac ctgctgacac cacccactgt tcgctgatga gggagccacc 420 gcagaagtgg tagccagaat tcaaggacac ctgntanggg acagaattct cctcacanat 480 gtanccccca acgatcttgc atcatcatca aagggggcag caacagcagc ttgcaacaaa 540 ggtaaggatc agaattagat tctgcc 566 <210> SEQ ID NO 101 <211> LENGTH: 743 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 35, 691, 736, 737 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 101 gcctgacgga actgccacga tgctgccact ttggnctctt tcactgctgc tgggagcagt 60 agcaggaaaa gaagtttgct acgaaagact cggctgcttc agtgatgact ccccatggtc 120 aggaattacg gaaagacccc tccatatatt gccttggtct ccaaaagatg tcaacacccg 180 cttcctccta tatactaatg agaacccaaa caactttcaa gaagttgccg cagattcatc 240 aagcatcagt ggctccaatt tcaaaacaaa tagaaaaact cgctttatta ttcatggatt 300 catagacaag ggagaagaaa actggctggc caatgtgtgc aagaatctgt tcaaggtgga 360 aagtgtgaac tgtatctgtg tggactggaa aggtggctcc cgaactggat acacacaagc 420 ctcgcagaac atcaggatcg tgggagcaga agtggcatat tttgttgaat ttcttcagtc 480 ggcgttcggt tactcacctt ccaacgtgca tgtcattggc cacagcctgg gtgcccacgc 540 tgctggggag gctggaagga gaaccaatgg gaccattgga cgcatcacag gggtggaccc 600 agcaagaacc tttctttcag ggcacacctg aattaagtcc gattggaccc caacgatgcc 660 aaaattgggg atgtaattca ccccggatgg ngcccccctt agtccccaat ttgggggttt 720 ggaatgaacc caatcnnggg gcc 743 <210> SEQ ID NO 102 <211> LENGTH: 782 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 60, 91, 94, 614, 656, 657, 665, 675, 703, 706, 718, 775, 781 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 102 tcccttccca gattccgtct atgtccacaa tctgagagag aatgttcttt ttacatccan 60 gcatttccac tcctccattt ggaaagaaat nggngggggc ccacgacttg gctcattcca 120 aaccccaaat tggggactat gggggcacca tccgtgtgaa ttacatccac aaatttggca 180 tcgctggggt ccaatcggac taattcaggt gtgccctgaa agcaaggttc tgctgggtcc 240 aaccctgtga tgcgtccaat ggtcccattg gttctccttc cagcctcccc agcagcgtgg 300 gcacccaggc tgtggccaat gacatgcacg ttggaaggtg agtaaccgaa cgccgactga 360 agaaattcaa caaaatatgc cacttctgct cccacgatcc tgatgttctg cgaggcttgt 420 gtgtatccag ttcgggagcc acctttccag tccacacaga tacagttcac actttccacc 480 ttgaacagat tcttgcacac attggccagc cagttttctt ctcccttgtc tatgaatcca 540 tgaataataa agcgagtttt tctattttgt tttgaaattg gagccacttg atgctttgat 600 gaaatcttgc gggnaacttc tttggaaaag gttggtttgg gggttcttca tttagnnatt 660 attanggaag gaaanccggg gtggttgaac attctttttt tgnaanaacc aagggcanat 720 aatattggga agggggggtc tttttcccgt aaattccctt gacccattgg ggggnagttc 780 na 782 <210> SEQ ID NO 103 <211> LENGTH: 712 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 38, 40, 650, 685, 687 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 103 gcctgacgga actgccacga tgctgccact ttgggctngn tcactgctgc tgggagcagt 60 agcaggaaaa gaagtttgct acgaaagact cggctgcttc agtgatgact ccccatggtc 120 aggaattacg gaaagacccc tccatatatt gccttggtct ccaaaagatg tcaacacccg 180 cttcctccta tatactaatg agaacccaaa caactttcaa gaagttgccg cagattcatc 240 aagcatcagt ggctccaatt tcaaaacaaa tagaaaaact cgctttatta ttcatggatt 300 catagacaag ggagaagaaa actggctggc caatgtgtgc aagaatctgt tcaaggtgga 360 aagtgtgaac tgtatctgtg tggactggaa aggtggctcc cgaactggat acacacaagc 420 ctcgcagaac atcaggatcg tgggagcaga agtggcatat tttgttgaat ttcttcagtc 480 ggcgttcggt tactcacctt ccaacgtgca tgtcattggc cacagcctgg gtgcccacgc 540 tgctggggag gctggaagga gaaccaatgg gaccattgga cgcatcacag gggtggaccc 600 acagaacctt gctttcaggg cacacctgaa ttagtccgat tggaccccan cgatgcccaa 660 tttgtggatg taaattcccc cggtngngcc ccctagtccc caatttgggg tt 712 <210> SEQ ID NO 104 <211> LENGTH: 872 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 60, 90, 91, 92, 649, 694, 703, 758, 781, 787, 802, 804, 810, 813, 814 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 104 tcccttccca gattccgtct atgtccacaa tctgagagag aatgttcttt ttacatccan 60 gcatttccac tcctccattt ggaaagaaan nnggggggcc cacgacttgg ctcattccaa 120 accccaaatt ggggactatg ggggcaccat ccgtgtgaat tacatccaca aatttggcat 180 cgctggggtc caatcggact aattcaggtg tgccctgaaa gcaaggttct gctgggtcca 240 accctgtgat gcgtccaatg gtcccattgg ttctccttcc agcctcccca gcagcgtggg 300 cacccaggct gtggccaatg acatgcacgt tggaaggtga gtaaccgaac gccgactgaa 360 gaaattcaac aaaatatgcc acttctgctc ccacgatcct gatgttctgc gaggcttgtg 420 tgtatccagt tcgggagcca cctttccagt ccacacagat acagttcaca ctttccacct 480 tgaacagatt cttgcacaca ttggccagcc agttttcttc tcccttgtct atgaatccat 540 gaataataaa gcgagttttt ctatttgttt tgaaattgga gccactgatg cttgatgaat 600 ctgcggcaac ttcttgaaag ttgtttgggt tctcattagt atataggang aagcgggtgt 660 tgacatcttt tggagaccaa ggcaatatat ggangggtct ttncgtaatt cctgaccatg 720 ggggagtcat cacttgaagc agcccgagtc tttcgtanca aacttctttt tctgctactg 780 nttccancaa cagtggaaag antnccaaan ggnnaagcat tctgggcaag ttcccgtcag 840 gcccaacaca atggaatttc cccccccttg ga 872 <210> SEQ ID NO 105 <211> LENGTH: 117 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 105 catggggagt catcactgaa gcagccgagt ctttcgtagc aaacttcttt tcctgctact 60 gctcccagca gcagtgaaag agtccaaagt ggcagcatcg tggcagttcc gtcaggc 117 <210> SEQ ID NO 106 <211> LENGTH: 117 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 106 gcctgacgga actgccacga tgctgccact ttggactctt tcactgctgc tgggagcagt 60 agcaggaaaa gaagtttgct acgaaagact cggctgcttc agtgatgact ccccatg 117 <210> SEQ ID NO 107 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 107 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 108 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 108 agtgggttac acaagctacg gcactttgga cagcaaataa gattgtttct gattatggaa 60 aggacccatc catcacttcc attctggacg ccctggatat cttcctcctg ccagtcacaa 120 accctgatgg atacgtgttc tctcaaacca aaaatcgtat gtggcggaag acccggtcca 180 aggtatctgg aagcctc 197 <210> SEQ ID NO 109 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 109 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt cctggagcag cagaataaga tcctgctggc cga 533 <210> SEQ ID NO 110 <211> LENGTH: 504 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 501, 503 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 110 tcggccagca ggatcttatt ctgctgctcc aggaagcgca ccttgtcgat gtagttggcg 60 aagcggtcat tcagctcctg cagctccacc ttctcgttgg tgcgggtgtt cttgaactcg 120 gtgttgatgg cgtcggccag cgagaagtcc accgagtcct gcaggagccg caccccgggc 180 acgctgctcc gcaggcgcac ggcagaggag cgcgtggcat acacgccgcc cggggacgag 240 gcgtagaggc tgcggctggt gctggggcgc agcgcgctgc ccaggctgta ggtgcgggtg 300 gacgtagtca cgtagctccg gctggagctc ggccggctcg cggtgcccgg gccgccgaac 360 atcctgcggt aggaggacga ggacacggac ctggtggaca tggctgcgga gggtggcgat 420 gggctgggcg gcggcggtgg cgcggactgg ctcccggaga agaggcgaac gagggcgcgg 480 tgggtgtggg tggtgggagc ncng 504 <210> SEQ ID NO 111 <211> LENGTH: 363 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 111 gctggacggc agctatgcga ctcaccgtgc tgtgtgctgt gtgcctgctg cctggcagcc 60 tggccctgcc gctgcctcag gaggcgggag gcatgagtga gctacagtgg gaacaggctc 120 aggactatct caagagattt tatctctatg actcagaaac aaaaaatgcc aacagtttag 180 aagccaaact caaggagatg caaaaattct ttggcctacc tataactgga atgttaaact 240 cccgcgtcat agaaataatg cagaagccca gatgtggagt gccagatgtt gcagaatact 300 cactatttcc aaatagccca aaatggactt ccaaagtggt cacctacagg atcgtatcat 360 ata 363 <210> SEQ ID NO 112 <211> LENGTH: 363 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 112 tatatgatac gatcctgtag gtgaccactt tggaagtcca ttttgggcta tttggaaata 60 gtgagtattc tgcaacatct ggcactccac atctgggctt ctgcattatt tctatgacgc 120 gggagtttaa cattccagtt ataggtaggc caaagaattt ttgcatctcc ttgagtttgg 180 cttctaaact gttggcattt tttgtttctg agtcatagag ataaaatctc ttgagatagt 240 cctgagcctg ttcccactgt agctcactca tgcctcccgc ctcctgaggc agcggcaggg 300 ccaggctgcc aggcagcagg cacacagcac acagcacggt gagtcgcata gctgccgtcc 360 agc 363 <210> SEQ ID NO 113 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 113 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt cctggagcag cagaataaga tcctgctggc cga 533 <210> SEQ ID NO 114 <211> LENGTH: 502 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 5, 46, 60, 64, 75, 97, 98, 99, 102, 105, 108, 110, 172, 216, 220, 231, 248, 472, 500 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 114 tcggncagca ggatcttatt ctgctgctcc aggaagcgca ccttgnctga tgtagttggn 60 gaancggtca ttcanctcct gcagctccac cttctcnnng gngcnggngn tcttgaactc 120 ggtgttgatg gctgtcggcc agcgagaagt ccaccgactc ctgcaagagc cncaccccgg 180 gcacgctgct ccgcaggcgc acggcagagg agcgcntggn atacacgccg nccggggacg 240 aggcgtanag gctgcggctg gtgctggggc gcagcgcgct gcccaggctg taggtgcggg 300 tggacgtagt cacgtagctc cggctggagc tcggccggct cgcggtgccc gggccgccga 360 acatcctgcg gtaggaggac gaggacacgg acctggtgga catggctgcg gagggtggcg 420 atgggctggg cggcggcggt ggcgcggact ggctcccgga gaagagcgaa cnaaggcgcc 480 gtgggtgtgg gtggtgggan cc 502 <210> SEQ ID NO 115 <211> LENGTH: 235 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 24, 32 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 115 gcacaaaact catgatgctc cggntgctca gntccctcct ccttgtggcc gttgcctcag 60 gctatggccc accttcctct cgcccttcca gccgcgttgt caatggtgag gatgcggtcc 120 cctacagctg gccctggcag gtttccctgc agtatgagaa aagtggaagc ttctaccaca 180 cgtgtggcgg tagcctcatc gcccccgatt gggttgtgac tgccggccac tgcat 235 <210> SEQ ID NO 116 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 116 ggaacaattg tctctggacg gcagctatgc gactcaccgt gctgtgtgct gtgtgcctgc 60 tgcctggcag cctggccctg ccgctgcctc aggaggcggg aggcatgagt gagctacagt 120 gggaacaggc tcaggactat ctcaagagat tttatctcta tgactcagaa acaaaaaatg 180 ccaacagttt agaagccaaa ctcaaggaga tgcaaaaatt ctttggccta cctataactg 240 gaatgttaaa ctcccgcgtc atagaaataa tgcagaagcc cagatgtgga gtgccagatg 300 ttgcagaata ctcactattt ccaaatagcc caaaatggac ttccaaagtg gtcacctaca 360 ggatcgtatc atata 375 <210> SEQ ID NO 117 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 117 tatatgatac gatcctgtag gtgaccactt tggaagtcca ttttgggcta tttggaaata 60 gtgagtattc tgcaacatct ggcactccac atctgggctt ctgcattatt tctatgacgc 120 gggagtttaa cattccagtt ataggtaggc caaagaattt ttgcatctcc ttgagtttgg 180 cttctaaact gttggcattt tttgtttctg agtcatagag ataaaatctc ttgagatagt 240 cctgagcctg ttcccactgt agctcactca tgcctcccgc ctcctgaggc agcggcaggg 300 ccaggctgcc aggcagcagg cacacagcac acagcacggt gagtcgcata gctgccgtcc 360 agagacaatt gttcc 375 <210> SEQ ID NO 118 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 118 ggctccggct gctcagttcc ctcctccttg tggccgttgc ctcaggctat ggcccacctt 60 cctctcgccc ttccagccgc gttgtcaatg gtgaggatgc ggtcccctac agctggccct 120 ggcaggtttc cctgcagtat gagaaaagtg gaagcttcta ccacacgtgt ggcggtagcc 180 tcatcgcccc cgattgggtt gtgactgccg gccactgcat 220 <210> SEQ ID NO 119 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 119 atgcagtggc cggcagtcac aacccaatcg ggggcgatga ggctaccgcc acacgtgtgg 60 tagaagcttc cacttttctc atactgcagg gaaacctgcc agggccagct gtaggggacc 120 gcatcctcac cattgacaac gcggctggaa gggcgagagg aaggtgggcc atagcctgag 180 gcaacggcca caaggaggag ggaactgagc agccggagcc 220 <210> SEQ ID NO 120 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 120 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 121 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 121 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 122 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 122 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 123 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 123 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 124 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 124 ggctccggct gctcagttcc ctcctccttg tggccgttgc ctcaggctat ggcccacctt 60 cctctcgccc ttccagccgc gttgtcaatg gtgaggatgc ggtcccctac agctggccct 120 ggcaggtttc cctgcagtat gagaaaagtg gaagcttcta ccacacgtgt ggcggtagcc 180 tcatcgcccc cgattgggtt gtgactgccg gccactgcat 220 <210> SEQ ID NO 125 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 125 atgcagtggc cggcagtcac aacccaatcg ggggcgatga ggctaccgcc acacgtgtgg 60 tagaagcttc cacttttctc atactgcagg gaaacctgcc agggccagct gtaggggacc 120 gcatcctcac cattgacaac gcggctggaa gggcgagagg aaggtgggcc atagcctgag 180 gcaacggcca caaggaggag ggaactgagc agccggagcc 220 <210> SEQ ID NO 126 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 126 ggctccggct gctcagttcc ctcctccttg tggccgttgc ctcaggctat ggcccacctt 60 cctctcgccc ttccagccgc gttgtcaatg gtgaggatgc ggtcccctac agctggccct 120 ggcaggtttc cctgcagtat gagaaaagtg gaagcttcta ccacacgtgt ggcggtagcc 180 tcatcgcccc cgattgggtt gtgactgccg gccactgcat 220 <210> SEQ ID NO 127 <211> LENGTH: 220 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 127 atgcagtggc cggcagtcac aacccaatcg ggggcgatga ggctaccgcc acacgtgtgg 60 tagaagcttc cacttttctc atactgcagg gaaacctgcc agggccagct gtaggggacc 120 gcatcctcac cattgacaac gcggctggaa gggcgagagg aaggtgggcc atagcctgag 180 gcaacggcca caaggaggag ggaactgagc agccggagcc 220 <210> SEQ ID NO 128 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 128 ggaacaattg tctctggacg gcagctatgc gactcaccgt gctgtgtgct gtgtgcctgc 60 tgcctggcag cctggccctg ccgctgcctc aggaggcggg aggcatgagt gagctacagt 120 gggaacaggc tcaggactat ctcaagagat tttatctcta tgactcagaa acaaaaaatg 180 ccaacagttt agaagccaaa ctcaaggaga tgcaaaaatt ctttggccta cctataactg 240 gaatgttaaa ctcccgcgtc atagaaataa tgcagaagcc cagatgtgga gtgccagatg 300 ttgcagaata ctcactattt ccaaatagcc caaaatggac ttccaaagtg gtcacctaca 360 ggatcgtatc atata 375 <210> SEQ ID NO 129 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 129 tatatgatac gatcctgtag gtgaccactt tggaagtcca ttttgggcta tttggaaata 60 gtgagtattc tgcaacatct ggcactccac atctgggctt ctgcattatt tctatgacgc 120 gggagtttaa cattccagtt ataggtaggc caaagaattt ttgcatctcc ttgagtttgg 180 cttctaaact gttggcattt tttgtttctg agtcatagag ataaaatctc ttgagatagt 240 cctgagcctg ttcccactgt agctcactca tgcctcccgc ctcctgaggc agcggcaggg 300 ccaggctgcc aggcagcagg cacacagcac acagcacggt gagtcgcata gctgccgtcc 360 agagacaatt gttcc 375 <210> SEQ ID NO 130 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 130 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt cctggagcag cagaataaga tcctgctggc cga 533 <210> SEQ ID NO 131 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 131 tcggccagca ggatcttatt ctgctgctcc aggaagcgca ccttgtcgat gtagttggcg 60 aagcggtcat tcagctcctg cagctccacc ttctcgttgg tgcgggtgtt cttgaactcg 120 gtgttgatgg cgtcggccag cgagaagtcc accgagtcct gcaggagccg caccccgggc 180 acgctgctcc gcaggcgcac ggcagaggag cgcgtggcat acacgccgcc cggggacgag 240 gcgtagaggc tgcggctggt gctggggcgc agcgcgctgc ccaggctgta ggtgcgggtg 300 gacgtagtca cgtagctccg gctggagctc ggccggctcg cggtgcccgg gccgccgaac 360 atcctgcggt aggaggacga ggacacggac ctggtggaca tggctgcgga gggtggcgat 420 gggctgggcg gcggcggtgg cgcggactgg ctcccggaga agaggcgaac gagggcgcgg 480 tgggtgtggg tggtgggagc gcggctgcgt ctctggcgcg gggacctcgg agc 533 <210> SEQ ID NO 132 <211> LENGTH: 532 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 132 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt ctggagcagc agaataagat cctgctggcc ga 532 <210> SEQ ID NO 133 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 5 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 133 tcggncagca ggatcttatt ctgctgctcc aggaagcgca ccttgtctat gtagttggcg 60 aagcggtcat tcagctcctg cagctccacc ttctcgttgg tgcgggtgtt cttgaactcg 120 gtgttgatgg cgtcggccag cgagaagtcc accgagtcct gcaggagccg caccccgggc 180 acgctgctcc gcaggcgcac ggcagaggag cgcgtggcat acacgccgcc cggggacgag 240 gcgtagaggc tgcggctggt gctggggcgc agcgcgctgc ccaggctgta ggtgcgggtg 300 gacgtagtca cgtagctccg gctggagctc ggccggctcg cggtgcccgg gccgccgaac 360 atcctgcggt aggaggacga ggacacggac ctggtggaca tggctgcgga gggtggcgat 420 gggctgggcg gcggcggtgg cgcggactgg ctcccggaga agaggcgaac gagggcgcgg 480 tgggtgtggg tggtgggagc gcggctgcgt ctctggcgcg gggacctcgg agc 533 <210> SEQ ID NO 134 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 134 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 135 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 135 agtgggttac acaagctacg gcactttgga cagcaaataa gattgtttct gattatggaa 60 aggacccatc catcacttcc attctggacg ccctggatat cttcctcctg ccagtcacaa 120 accctgatgg atacgtgttc tctcaaacca aaaatcgtat gtggcggaag acccggtcca 180 aggtatctgg aagcctc 197 <210> SEQ ID NO 136 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 136 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 137 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 137 agtgggttac acaagctacg gcactttgga cagcaaataa gattgtttct gattatggaa 60 aggacccatc catcacttcc attctggacg ccctggatat cttcctcctg ccagtcacaa 120 accctgatgg atacgtgttc tctcaaacca aaaatcgtat gtggcggaag acccggtcca 180 aggtatctgg aagcctc 197 <210> SEQ ID NO 138 <211> LENGTH: 197 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 138 gaggcttcca gataccttgg accgggtctt ccgccacata cgatttttgg tttgagagaa 60 cacgtatcca tcagggtttg tgactggcag gaggaagata tccagggcgt ccagaatgga 120 agtgatggat gggtcctttc cataatcaga aacaatctta tttgctgtcc aaagtgccgt 180 agcttgtgta acccact 197 <210> SEQ ID NO 139 <211> LENGTH: 938 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 8, 16, 919, 929 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 139 taagaaanta rgggrnttcc cccctaagtc ccaagggggg ggaaattcca ttggttgkgc 60 ctgacggaac tgccacgatg ctgcccactt tggactcttt cactgctgst gggagcagta 120 gcaggaaaag aagtttgcta cgaaagactc ggctgcttca gtgatgactc cccatggtca 180 ggaattacgg aaagacccct ccatatattg ccttggtctc caaaagatgt caacacccgc 240 ttcctcctat atactaatga gaacccaaac aactttcaag aagttgccgc agattcatca 300 agcatcagtg gctccaattt caaaacaaat agaaaaactc gctttattat tcatggattc 360 atagacaagg gagaagaaaa ctggctggcc aatgtgtgca agaatctgtt caaggtggaa 420 agtgtgaact gtatctgtgt ggactggaaa ggtggctccc gaactggata cacacaagcc 480 tcgcagaaca tcaggatcgt gggagcagaa gtggcatatt ttgttgaatt tcttcagtcg 540 gcgttcggtt actcaccttc caacgtgcat gtcattggcc acagcctggg tgcccacgct 600 gctggggagg ctggaaggag aaccaatggg accattggac gcatcacagg gttggaccca 660 gcagaacctt gctttcaggg cacacctgaa ttagtccgat tggaccccag cgatgccaaa 720 tttgtggatg taattcacac ggatggtgcc cccatagtcc ccaatttggg gtttggaatg 780 agccaastcg tgggccacct agatttcttt ccaaatggag gagtggaaat gcctggatgt 840 aaaaagaaca ttctctctca gattgtggac atagacggaa tctgggaagg gatttttttc 900 aaaaaagggg gggaccaana aaaaggaana ttttgggg 938 <210> SEQ ID NO 140 <211> LENGTH: 256 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 140 ttggtccggc cccccctcga gatgcagtgg ccggcagtca caacccaatc gggggcgatg 60 aggctaccgc cacacgtgtg gtagaagctt ccacttttct catactgcag ggaaacctgc 120 cagggccagc tgtaggggac cgcatcctca ccattgacaa cgcggctgga agggcgagag 180 gaaggtgggc catagcctga ggcaacggcc acaaggagga gggaactgag cagccggagc 240 ctcatgagtt ttgtgc 256 <210> SEQ ID NO 141 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141 tatatgatac gatcctgtag gtgaccactt tggaagtcca ttttgggcta tttggaaata 60 gtgagtattc tgcaacatct ggcactccac atctgggctt ctgcattatt tctatgacgc 120 gggagtttaa cattccagtt ataggtaggc caaagaattt ttgcatctcc ttgagtttgg 180 cttctaaact gttggcattt tttgtttctg agtcatagag ataaaatctc ttgagatagt 240 cctgagcctg ttcccactgt agctcactca tgcctcccgc ctcctgaggc agcggcaggg 300 ccaggctgcc aggcagcagg cacacagcac acagcacggt gagtcgcata gctgccgtcc 360 agagacaatt gttcc 375 <210> SEQ ID NO 142 <211> LENGTH: 567 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 142 gaagcccaca cagaacatgt tgttggtaat ctttccaggg taggaggctt cacactcagc 60 ctggctcagc acaggagcat ccaggcactg cagctcgtct gggtagtcgg caccagaact 120 cagagtgttg ccccagccgg agatgaggga ctcggtgcca gcagctggag gggcagtggg 180 cagagagatg gcggacacgc gggaattgat gacggcaggt gaggagagct tgatcagcag 240 gatgtcattg tccagagtcc ggctgttgta tttggggtgg cggatgatct tggctgcatt 300 gatgaactgt tcattcccct ccaggacttc gatgttgtgc tctcccagtc tcacctggat 360 gcgggacttg tagcagtgac ctgctgacac cacccactgt tcgctgatga gggagccacc 420 gcagaagtgg tagccagaat tcaaggacac ctggtagggg acagaattct cctcacagat 480 gtagccccca acgatcttgt catcatcatc aaagggggca gcaacagcag ctgcaacaaa 540 ggtaaggatc agaagtagat tcatgcc 567 <210> SEQ ID NO 143 <211> LENGTH: 533 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 143 gctccgaggt ccccgcgcca gagacgcagc cgcgctccca ccacccacac ccaccgcgcc 60 ctcgttcgcc tcttctccgg gagccagtcc gcgccaccgc cgccgcccag cccatcgcca 120 ccctccgcag ccatgtccac caggtccgtg tcctcgtcct cctaccgcag gatgttcggc 180 ggcccgggca ccgcgagccg gccgagctcc agccggagct acgtgactac gtccacccgc 240 acctacagcc tgggcagcgc gctgcgcccc agcaccagcc gcagcctcta cgcctcgtcc 300 ccgggcggcg tgtatgccac gcgctcctct gccgtgcgcc tgcggagcag cgtgcccggg 360 gtgcggctcc tgcaggactc ggtggacttc tcgctggccg acgccatcaa caccgagttc 420 aagaacaccc gcaccaacga gaaggtggag ctgcaggagc tgaatgaccg cttcgccaac 480 tacatcgaca aggtgcgctt cctggagcag cagaataaga tcctgctggc cga 533 <210> SEQ ID NO 144 <211> LENGTH: 1306 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 144 ggccatgagg ttgatcctgt tttttggtgc cctttttggg catatctact gtctagaaac 60 atttgtggga gaccaagttc ttgagattgt accaagcaat gaagaacaaa ttaaaaatct 120 gctacaattg gaggctcaag aacatctcca gcttgatttt tggaaatcac ccaccacccc 180 aggggagaca gcccacgtcc gagttccctt cgtcaacgtc caggcagtca aagtgttctt 240 ggagtcccag ggaattgcct attccatcat gattgaagac gtgcaggtcc tgttggacaa 300 agagaatgaa gaaatgcttt ttaataggag aagagaacgg agtggtaact tcaattttgg 360 ggcctaccat accctggaag agatttccca agaaatggat aacctcgtgg ctgagcaccc 420 tggtctagtg agcaaagtga atattggctc ttcttttgag aaccggccta tgaacgtgct 480 caagttcagc accggaggag acaagccagc tatctggctg gatgctggga tccatgctcg 540 agagtgggtt acacaagcta cggcactttg gacagcaaat aagattgttt ctgattatgg 600 aaaggaccca tccatcactt ccattctgga cgccctggat atcttcctcc tgccagtcac 660 aaaccctgat ggatacgtgt tctctcaaac caaaaatcgt atgtggcgga agacccggtc 720 caaggtatct ggaagcctct gtgttggtgt ggatcctaac cggaactggg atgcaggttt 780 tggaggacct ggagccagca gcaacccttg ctctgattca taccacggac ccagtgccaa 840 ctctgaagtt gaagtgaaat ccatagtgga cttcatcaag agtcatggaa aagtcaaggc 900 cttcattatc ctccacagct attcccagct gctgatgttc ccctatgggt acaaatgtac 960 caagttagat gactttgatg agctgagtga agtggcccaa aaggctgccc aatctctgag 1020 aagcctgcat ggcaccaagt acaaagtggg accaatctgc tctgtcatct accaagccag 1080 tggaggaagc attgactggt cctatgatta tggcatcaag tactcatttg cctttgaact 1140 gagagacaca gggcgctacg gcttcctctt gccagcccgt cagatcctgc ccacagccga 1200 ggagacctgg cttggcttga aggcaatcat ggagcatgtg cgagaccacc cctattaggg 1260 ccctggggaa gaaacaagag ccattaaaat ctctttggtt tgaagc 1306 <210> SEQ ID NO 145 <211> LENGTH: 1471 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 145 ggaactgcca cgatgctgcc actttggact ctttcactgc tgctgggagc agtagcagga 60 aaagaagttt gctacgaaag actcggctgc ttcagtgatg actccccatg gtcaggaatt 120 acggaaagac ccctccatat attgccttgg tctccaaaag atgtcaacac ccgcttcctc 180 ctatatacta atgagaaccc aaacaacttt caagaagttg ccgcagattc atcaagcatc 240 agtggctcca atttcaaaac aaatagaaaa actcgcttta ttattcatgg attcatagac 300 aagggagaag aaaactggct ggccaatgtg tgcaagaatc tgttcaaggt ggaaagtgtg 360 aactgtatct gtgtggactg gaaaggtggc tcccgaactg gatacacaca agcctcgcag 420 aacatcagga tcgtgggagc agaagtggca tattttgttg aatttcttca gtcggcgttc 480 ggttactcac cttccaacgt gcatgtcatt ggccacagcc tgggtgccca cgctgctggg 540 gaggctggaa ggagaaccaa tgggaccatt ggacgcatca cagggttgga cccagcagaa 600 ccttgctttc agggcacacc tgaattagtc cgattggacc ccagcgatgc caaatttgtg 660 gatgtaattc acacggatgg tgcccccata gtccccaatt tggggtttgg aatgagccaa 720 gtcgtgggcc acctagattt ctttccaaat ggaggagtgg aaatgcctgg atgtaaaaag 780 aacattctct ctcagattgt ggacatagac ggaatctggg aagggactcg agactttgcg 840 gcctgtaatc acttaagaag ctacaaatat tacactgata gcatcgtcaa ccctgatggc 900 tttgctggat tcccctgtgc ctcttacaac gtcttcactg caaacaagtg tttcccttgt 960 ccaagtggag gctgcccaca gatgggtcac tatgctgata gatatcctgg gaaaacaaat 1020 gatgtgggcc agaaatttta tctagacact ggtgatgcca gtaattttgc acgttggagg 1080 tataaggtat ctgtcacact gtctggaaaa aaggttacag gacacatact agtttctttg 1140 ttcggaaata aaggaaactc taagcagtat gaaattttca agggcactct caaaccagat 1200 agtactcatt ccaatgaatt tgactcagat gtggatgttg gggacttgca gatggttaaa 1260 tttatttggt ataacaatgt gatcaaccca actttaccta gagtgggagc atccaagatt 1320 atagtggaga caaatgttgg aaaacagttc aacttctgta gtccagaaac cgtcagggag 1380 gaagttctgc tcaccctcac accgtgttag gagactactg ttatttgacc aatgaattga 1440 cttctaataa aatctagtgg tgatgcaaaa a 1471 <210> SEQ ID NO 146 <211> LENGTH: 897 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 146 cgtcctacat tcacaaaact catgatgctc cggctgctca gttccctcct ccttgtggcc 60 gttgcctcag gctatggccc accttcctct cactcttcca gccgcgttgt ccatggtgag 120 gatgcggtcc cctacagctg gccctggcag gtttccctgc agtatgagaa aagtggaagc 180 ttctaccaca cgtgtggcgg tagcctcatc gcccccgatt gggttgtgac tgccggccac 240 tgcatctcga gggatctgac ctaccaggtg gtgttgggtg agtacaacct tgctgtgaag 300 gagggccccg agcaggtgat ccccatcaac tctgaggagc tgtttgtgca tccactctgg 360 aaccgctcgt gtgtggcctg tggcaatgac atcgccctca tcaagctctc acgcagcgcc 420 cagctgggag atgccgtcca gctcgcctca ctccctcccg ctggtgacat ccttcccaac 480 aagacaccct gctacatcac cggctggggc cgtctctata ccaatgggcc actcccagac 540 aagctgcagc aggcccggct gcccgtggtg gactataagc actgctccag gtggaactgg 600 tggggttcca ccgtgaagaa aaccatggtg tgtgctggag ggtacatccg ctccggctgc 660 aacggtgact ctggaggacc cctcaactgc cccacagagg atggtggctg gcaggtccac 720 ggtgtgacca gctttgtttc tggctttggc tgcaacttca tctggaagcc tacagtgttc 780 actcgagtct ccgccttcat cgactggatt gaggagacca tagcaagcca ctagagccaa 840 ggcccagctg gcagtgctga cgatccccac atcctgaata aagaataaag atctctc 897 <210> SEQ ID NO 147 <211> LENGTH: 1078 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 147 aagaacaatt gtctctggac ggcagctatg cgactcaccg tgctgtgtgc tgtgtgcctg 60 ctgcctggca gcctggccct gccgctgcct caggaggcgg gaggcatgag tgagctacag 120 tgggaacagg ctcaggacta tctcaagaga ttttatctct atgactcaga aacaaaaaat 180 gccaacagtt tagaagccaa actcaaggag atgcaaaaat tctttggcct acctataact 240 ggaatgttaa actcccgcgt catagaaata atgcagaagc ccagatgtgg agtgccagat 300 gttgcagaat actcactatt tccaaatagc ccaaaatgga cttccaaagt ggtcacctac 360 aggatcgtat catatactcg agacttaccg catattacag tggatcgatt agtgtcaaag 420 gctttaaaca tgtggggcaa agagatcccc ctgcatttca ggaaagttgt atggggaact 480 gctgacatca tgattggctt tgcgcgagga gctcatgggg actcctaccc atttgatggg 540 ccaggaaaca cgctggctca tgcctttgcg cctgggacag gtctcggagg agatgctcac 600 ttcgatgagg atgaacgctg gacggatggt agcagtctag ggattaactt cctgtatgct 660 gcaactcatg aacttggcca ttctttgggt atgggacatt cctctgatcc taatgcagtg 720 atgtatccaa cctatggaaa tggagatccc caaaatttta aactttccca ggatgatatt 780 aaaggcattc agaaactata tggaaagaga agtaattcaa gaaagaaata gaaacttcag 840 gcagaacatc cattcattca ttcattggat tgtatatcat tgttgcacaa tcagaattga 900 taagcactgt tcctccactc catttagcaa ttatgtcacc cttttttatt gcagttggtt 960 tttgaatgtc tttcactcct tttattggtt aaactccttt atggtgtgac tgtgtcttat 1020 tccatctatg agctttgtca gtgcgcgtag atgtcaataa atgttacata cacaaata 1078 <210> SEQ ID NO 148 <211> LENGTH: 802 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 148 aacaccatga atctactcct gatccttacc tttgttgcag ctgctgttgc tgcccccttt 60 gatgatgatg acaagatcgt tgggggctac atctgtgagg agaattctgt cccctaccag 120 gtgtccttga attctggcta ccacttctgc ggtggctccc tcatcagcga acagtgggtg 180 gtgtcagcag gtcactgcta caagtcccgc atccaggtga gactgggaga gcacaacatc 240 gaagtcctgg aggggaatga acagttcatc aatgcggcca agatcatccg ccaccccaaa 300 tacaacagcc ggactctgga caatgacatc ctgctgatca agctctcctc acctgccgtc 360 atcaattccc gcgtgtccgc catctctctg cccactgccc ctccagctgc tggcaccgag 420 tccctcatct ccggctgggg caacactctg agttctggtg ccgactaccc agacgagctg 480 cagtgcctgg atgctcctgt gctgagccag gctgagtgtg aagcctccta ccctggaaag 540 attaccaaca acatgttctg tgtgggcttc ctcgagggag gcaaggattc ctgccagggt 600 gattctggtg gccctgtggt ctccaatgga gagctccaag gaattgtctc ctggggctat 660 ggctgtgccc agaagaacag gcctggagtc tacaccaagg tctacaacta tgtggactgg 720 attaaggaca ccatagctgc caacagctaa agcccccagt ccctctgcag tctctatacc 780 aataaagtga ccctgctctc ac 802 <210> SEQ ID NO 149 <211> LENGTH: 1749 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 149 gagggcgccc ccaccccacc cgcccaccct ccccgcttct cgctaggtcc cgattggctg 60 gcgcgctccg cggctgggat ggcagtggga ggggaccctc tttcctaacg gggttataaa 120 aacagcgccc tcggcggggt ccagtcctct gccactctcg ctccgaggtc cccgcgccag 180 agacgcagcc gcgctcccac cacccacacc caccgcgccc tcgttcgcct cttctccggg 240 agccagtccg cgccaccgcc gccgcccagc ccatcgccac cctccgcagc catgtccacc 300 aggtccgtgt cctcgtcctc ctaccgcagg atgttcggcg gcccgggcac cgcgagccgg 360 ccgagctcca gccggagcta cgtgactacg tccacccgca cctacagcct gggcgacgcg 420 ctgcgcccca gcaccagccg cagcctctac gcctcgtccc cgggcggcgt gtatgccacg 480 cgctcctctg ccgtgcgcct gcggagcagc gtgcccgggg tgcggctcct gcaggactcg 540 gtggacttct cgctggccga cgccatcaac accgagttca agaacacccg caccaacgag 600 aaggtggagc tgcaggagct gaatgaccgc ttcgccaact acatcgacaa ggtgcgcttc 660 ctggagcagc agaataagat cctgctggcc gagctcgagc agctcaaggg ccaaggcaag 720 tcgcgcctag gggacctcta cgaggaggag atgcgggagc tgcgccggca ggtggaccag 780 ctaaccaacg acaaagcccg cgtcgaggtg gagcgcgaca acctggccga ggacatcatg 840 cgcctccggg aaaaattgca ggaggagatg cttcagagag aggaagccga aaacaccctg 900 caatctttca gacaggatgt tgacaatgcg tctctggcac gtcttgacct tgaacgcaaa 960 gtggaatctt tgcaagaaga gattgccttt ttgaagaaac tccacgaaga ggaaatccag 1020 gagctgcagg ctcagattca ggaacagcat gtccaaatcg atgtggatgt ttccaagcct 1080 gacctcacgg ctgccctgcg tgacgtacgt cagcaatatg aaagtgtggc tgccaagaac 1140 ctgcaggagg cagaagaatg gtacaaatcc aagtttgctg acctctctga ggctgccaac 1200 cggaacaatg acgccctgcg ccaggcaaag caggagtcca ctgagtaccg gagacaggtg 1260 cagtccctca cctgtgaagt ggatgccctt aaaggaacca atgagtccct ggaacgccag 1320 atgcgtgaaa tggaagagaa ctttgccgtt gaagctgcta actaccaaga cactattggc 1380 cgcctgcagg atgagattca gaatatgaag gaggaaatgg ctcgtcacct tcgtgaatac 1440 caagacctgc tcaatgttaa gatggccctt gacattgaga ttgccaccta caggaagctg 1500 ctggaaggcg aggagagcag gatttctctg cctcttccaa acttttcctc cctgaacctg 1560 agggaaacta atctggattc actccctctg gttgataccc actcaaaaag gacattcctg 1620 attaagacgg ttgaaactag agatggacag gttatcaacg aaacttctca gcatcacgat 1680 gaccttgaat aaacaattgc acactcagtg cagcactcat ataccagcag ataaaagaat 1740 ccatatctt 1749 <210> SEQ ID NO 150 <211> LENGTH: 417 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 150 Met Arg Leu Ile Leu Phe Phe Gly Ala Leu Phe Gly His Ile Tyr Cys 5 10 15 Leu Glu Thr Phe Val Gly Asp Gln Val Leu Glu Ile Val Pro Ser Asn 20 25 30 Glu Glu Gln Ile Lys Asn Leu Leu Gln Leu Glu Ala Gln Glu His Leu 35 40 45 Gln Leu Asp Phe Trp Lys Ser Pro Thr Thr Pro Gly Glu Thr Ala His 50 55 60 Val Arg Val Pro Phe Val Asn Val Gln Ala Val Lys Val Phe Leu Glu 65 70 75 80 Ser Gln Gly Ile Ala Tyr Ser Ile Met Ile Glu Asp Val Gln Val Leu 85 90 95 Leu Asp Lys Glu Asn Glu Glu Met Leu Phe Asn Arg Arg Arg Glu Arg 100 105 110 Ser Gly Asn Phe Asn Phe Gly Ala Tyr His Thr Leu Glu Glu Ile Ser 115 120 125 Gln Glu Met Asp Asn Leu Val Ala Glu His Pro Gly Leu Val Ser Lys 130 135 140 Val Asn Ile Gly Ser Ser Phe Glu Asn Arg Pro Met Asn Val Leu Lys 145 150 155 160 Phe Ser Thr Gly Gly Asp Lys Pro Ala Ile Trp Leu Asp Ala Gly Ile 165 170 175 His Ala Arg Glu Trp Val Thr Gln Ala Thr Ala Leu Trp Thr Ala Asn 180 185 190 Lys Ile Val Ser Asp Tyr Gly Lys Asp Pro Ser Ile Thr Ser Ile Leu 195 200 205 Asp Ala Leu Asp Ile Phe Leu Leu Pro Val Thr Asn Pro Asp Gly Tyr 210 215 220 Val Phe Ser Gln Thr Lys Asn Arg Met Trp Arg Lys Thr Arg Ser Lys 225 230 235 240 Val Ser Gly Ser Leu Cys Val Gly Val Asp Pro Asn Arg Asn Trp Asp 245 250 255 Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser Asn Pro Cys Ser Asp Ser 260 265 270 Tyr His Gly Pro Ser Ala Asn Ser Glu Val Glu Val Lys Ser Ile Val 275 280 285 Asp Phe Ile Lys Ser His Gly Lys Val Lys Ala Phe Ile Ile Leu His 290 295 300 Ser Tyr Ser Gln Leu Leu Met Phe Pro Tyr Gly Tyr Lys Cys Thr Lys 305 310 315 320 Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Ala Gln Lys Ala Ala Gln 325 330 335 Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Lys Val Gly Pro Ile Cys 340 345 350 Ser Val Ile Tyr Gln Ala Ser Gly Gly Ser Ile Asp Trp Ser Tyr Asp 355 360 365 Tyr Gly Ile Lys Tyr Ser Phe Ala Phe Glu Leu Arg Asp Thr Gly Arg 370 375 380 Tyr Gly Phe Leu Leu Pro Ala Arg Gln Ile Leu Pro Thr Ala Glu Glu 385 390 395 400 Thr Trp Leu Gly Leu Lys Ala Ile Met Glu His Val Arg Asp His Pro 405 410 415 Tyr <210> SEQ ID NO 151 <211> LENGTH: 465 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 151 Met Leu Pro Leu Trp Thr Leu Ser Leu Leu Leu Gly Ala Val Ala Gly 5 10 15 Lys Glu Val Cys Tyr Glu Arg Leu Gly Cys Phe Ser Asp Asp Ser Pro 20 25 30 Trp Ser Gly Ile Thr Glu Arg Pro Leu His Ile Leu Pro Trp Ser Pro 35 40 45 Lys Asp Val Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro Asn 50 55 60 Asn Phe Gln Glu Val Ala Ala Asp Ser Ser Ser Ile Ser Gly Ser Asn 65 70 75 80 Phe Lys Thr Asn Arg Lys Thr Arg Phe Ile Ile His Gly Phe Ile Asp 85 90 95 Lys Gly Glu Glu Asn Trp Leu Ala Asn Val Cys Lys Asn Leu Phe Lys 100 105 110 Val Glu Ser Val Asn Cys Ile Cys Val Asp Trp Lys Gly Gly Ser Arg 115 120 125 Thr Gly Tyr Thr Gln Ala Ser Gln Asn Ile Arg Ile Val Gly Ala Glu 130 135 140 Val Ala Tyr Phe Val Glu Phe Leu Gln Ser Ala Phe Gly Tyr Ser Pro 145 150 155 160 Ser Asn Val His Val Ile Gly His Ser Leu Gly Ala His Ala Ala Gly 165 170 175 Glu Ala Gly Arg Arg Thr Asn Gly Thr Ile Gly Arg Ile Thr Gly Leu 180 185 190 Asp Pro Ala Glu Pro Cys Phe Gln Gly Thr Pro Glu Leu Val Arg Leu 195 200 205 Asp Pro Ser Asp Ala Lys Phe Val Asp Val Ile His Thr Asp Gly Ala 210 215 220 Pro Ile Val Pro Asn Leu Gly Phe Gly Met Ser Gln Val Val Gly His 225 230 235 240 Leu Asp Phe Phe Pro Asn Gly Gly Val Glu Met Pro Gly Cys Lys Lys 245 250 255 Asn Ile Leu Ser Gln Ile Val Asp Ile Asp Gly Ile Trp Glu Gly Thr 260 265 270 Arg Asp Phe Ala Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr Thr 275 280 285 Asp Ser Ile Val Asn Pro Asp Gly Phe Ala Gly Phe Pro Cys Ala Ser 290 295 300 Tyr Asn Val Phe Thr Ala Asn Lys Cys Phe Pro Cys Pro Ser Gly Gly 305 310 315 320 Cys Pro Gln Met Gly His Tyr Ala Asp Arg Tyr Pro Gly Lys Thr Asn 325 330 335 Asp Val Gly Gln Lys Phe Tyr Leu Asp Thr Gly Asp Ala Ser Asn Phe 340 345 350 Ala Arg Trp Arg Tyr Lys Val Ser Val Thr Leu Ser Gly Lys Lys Val 355 360 365 Thr Gly His Ile Leu Val Ser Leu Phe Gly Asn Lys Gly Asn Ser Lys 370 375 380 Gln Tyr Glu Ile Phe Lys Gly Thr Leu Lys Pro Asp Ser Thr His Ser 385 390 395 400 Asn Glu Phe Asp Ser Asp Val Asp Val Gly Asp Leu Gln Met Val Lys 405 410 415 Phe Ile Trp Tyr Asn Asn Val Ile Asn Pro Thr Leu Pro Arg Val Gly 420 425 430 Ala Ser Lys Ile Ile Val Glu Thr Asn Val Gly Lys Gln Phe Asn Phe 435 440 445 Cys Ser Pro Glu Thr Val Arg Glu Glu Val Leu Leu Thr Leu Thr Pro 450 455 460 Cys 465 <210> SEQ ID NO 152 <211> LENGTH: 270 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 152 Met Met Leu Arg Leu Leu Ser Ser Leu Leu Leu Val Ala Val Ala Ser 5 10 15 Gly Tyr Gly Pro Pro Ser Ser His Ser Ser Ser Arg Val Val His Gly 20 25 30 Glu Asp Ala Val Pro Tyr Ser Trp Pro Trp Gln Val Ser Leu Gln Tyr 35 40 45 Glu Lys Ser Gly Ser Phe Tyr His Thr Cys Gly Gly Ser Leu Ile Ala 50 55 60 Pro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser Arg Asp Leu Thr 65 70 75 80 Tyr Gln Val Val Leu Gly Glu Tyr Asn Leu Ala Val Lys Glu Gly Pro 85 90 95 Glu Gln Val Ile Pro Ile Asn Ser Glu Glu Leu Phe Val His Pro Leu 100 105 110 Trp Asn Arg Ser Cys Val Ala Cys Gly Asn Asp Ile Ala Leu Ile Lys 115 120 125 Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Val Gln Leu Ala Ser Leu 130 135 140 Pro Pro Ala Gly Asp Ile Leu Pro Asn Lys Thr Pro Cys Tyr Ile Thr 145 150 155 160 Gly Trp Gly Arg Leu Tyr Thr Asn Gly Pro Leu Pro Asp Lys Leu Gln 165 170 175 Gln Ala Arg Leu Pro Val Val Asp Tyr Lys His Cys Ser Arg Trp Asn 180 185 190 Trp Trp Gly Ser Thr Val Lys Lys Thr Met Val Cys Ala Gly Gly Tyr 195 200 205 Ile Arg Ser Gly Cys Asn Gly Asp Ser Gly Gly Pro Leu Asn Cys Pro 210 215 220 Thr Glu Asp Gly Gly Trp Gln Val His Gly Val Thr Ser Phe Val Ser 225 230 235 240 Gly Phe Gly Cys Asn Phe Ile Trp Lys Pro Thr Val Phe Thr Arg Val 245 250 255 Ser Ala Phe Ile Asp Trp Ile Glu Glu Thr Ile Ala Ser His 260 265 270 <210> SEQ ID NO 153 <211> LENGTH: 267 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 153 Met Arg Leu Thr Val Leu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu 5 10 15 Ala Leu Pro Leu Pro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln Trp 20 25 30 Glu Gln Ala Gln Asp Tyr Leu Lys Arg Phe Tyr Leu Tyr Asp Ser Glu 35 40 45 Thr Lys Asn Ala Asn Ser Leu Glu Ala Lys Leu Lys Glu Met Gln Lys 50 55 60 Phe Phe Gly Leu Pro Ile Thr Gly Met Leu Asn Ser Arg Val Ile Glu 65 70 75 80 Ile Met Gln Lys Pro Arg Cys Gly Val Pro Asp Val Ala Glu Tyr Ser 85 90 95 Leu Phe Pro Asn Ser Pro Lys Trp Thr Ser Lys Val Val Thr Tyr Arg 100 105 110 Ile Val Ser Tyr Thr Arg Asp Leu Pro His Ile Thr Val Asp Arg Leu 115 120 125 Val Ser Lys Ala Leu Asn Met Trp Gly Lys Glu Ile Pro Leu His Phe 130 135 140 Arg Lys Val Val Trp Gly Thr Ala Asp Ile Met Ile Gly Phe Ala Arg 145 150 155 160 Gly Ala His Gly Asp Ser Tyr Pro Phe Asp Gly Pro Gly Asn Thr Leu 165 170 175 Ala His Ala Phe Ala Pro Gly Thr Gly Leu Gly Gly Asp Ala His Phe 180 185 190 Asp Glu Asp Glu Arg Trp Thr Asp Gly Ser Ser Leu Gly Ile Asn Phe 195 200 205 Leu Tyr Ala Ala Thr His Glu Leu Gly His Ser Leu Gly Met Gly His 210 215 220 Ser Ser Asp Pro Asn Ala Val Met Tyr Pro Thr Tyr Gly Asn Gly Asp 225 230 235 240 Pro Gln Asn Phe Lys Leu Ser Gln Asp Asp Ile Lys Gly Ile Gln Lys 245 250 255 Leu Tyr Gly Lys Arg Ser Asn Ser Arg Lys Lys 260 265 <210> SEQ ID NO 154 <211> LENGTH: 247 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 154 Met Asn Leu Leu Leu Ile Leu Thr Phe Val Ala Ala Ala Val Ala Ala 5 10 15 Pro Phe Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Ile Cys Glu Glu 20 25 30 Asn Ser Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe Cys 35 40 45 Gly Gly Ser Leu Ile Ser Glu Gln Trp Val Val Ser Ala Gly His Cys 50 55 60 Tyr Lys Ser Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Glu Val 65 70 75 80 Leu Glu Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys Ile Ile Arg His 85 90 95 Pro Lys Tyr Asn Ser Arg Thr Leu Asp Asn Asp Ile Leu Leu Ile Lys 100 105 110 Leu Ser Ser Pro Ala Val Ile Asn Ser Arg Val Ser Ala Ile Ser Leu 115 120 125 Pro Thr Ala Pro Pro Ala Ala Gly Thr Glu Ser Leu Ile Ser Gly Trp 130 135 140 Gly Asn Thr Leu Ser Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln Cys 145 150 155 160 Leu Asp Ala Pro Val Leu Ser Gln Ala Glu Cys Glu Ala Ser Tyr Pro 165 170 175 Gly Lys Ile Thr Asn Asn Met Phe Cys Val Gly Phe Leu Glu Gly Gly 180 185 190 Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Ser Asn Gly 195 200 205 Glu Leu Gln Gly Ile Val Ser Trp Gly Tyr Gly Cys Ala Gln Lys Asn 210 215 220 Arg Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val Asp Trp Ile Lys 225 230 235 240 Asp Thr Ile Ala Ala Asn Ser 245 <210> SEQ ID NO 155 <211> LENGTH: 466 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 155 Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly 5 10 15 Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20 25 30 Thr Ser Thr Arg Thr Tyr Ser Leu Gly Asp Ala Leu Arg Pro Ser Thr 35 40 45 Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55 60 Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu 65 70 75 80 Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95 Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110 Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120 125 Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130 135 140 Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln 145 150 155 160 Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp 165 170 175 Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu 180 185 190 Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln 195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val 210 215 220 Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu 225 230 235 240 Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile 245 250 255 Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270 Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285 Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300 Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg 305 310 315 320 Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335 Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350 Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360 365 Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370 375 380 Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr 385 390 395 400 Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405 410 415 Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro 420 425 430 Leu Val Asp Thr His Ser Lys Arg Thr Phe Leu Ile Lys Thr Val Glu 435 440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp 450 455 460 Leu Glu 465 

What is claimed:
 1. An isolated polynucleotide comprising a sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO:1-149; (b) complements of the sequences provided in SEQ ID NO:1-149; (c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NO:1-149; (d) sequences that hybridize to a sequence provided in SEQ ID NO:1-149, under moderately stringent conditions; (e) sequences having at least 75% identity to a sequence of SEQ ID NO:1-149; (f) sequences having at least 90% identity to a sequence of SEQ ID NO:1-149; and (g) degenerate variants of a sequence provided in SEQ ID NO:1-149.
 2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences encoded by a polynucleotide of claim 1; and (b) sequences having at least 70% identity to a sequence encoded by a polynucleotide of claim 1; (c) sequences having at least 90% identity to a sequence encoded by a polynucleotide of claim 1; (d) sequences provided by SEQ ID NO:150-155; (e) sequences having at least 70% identity to a sequence provided by SEQ ID NO:150-155; and (f) sequences having at least 90% identity to a sequence provided by SEQ ID NO:150-155.
 3. An expression vector comprising a polynucleotide of claim 1 operably linked to an expression control sequence.
 4. A host cell transformed or transfected with an expression vector according to claim
 3. 5. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide of claim
 2. 6. A method for detecting the presence of a cancer in a patient, comprising the steps of: (a) obtaining a biological sample from the patient; (b) contacting the biological sample with a binding agent that binds to a polypeptide of claim 2; (c) detecting in the sample an amount of polypeptide that binds to the binding agent; and (d) comparing the amount of polypeptide to a predetermined cut-off value and therefrom determining the presence of a cancer in the patient.
 7. A fusion protein comprising at least one polypeptide according to claim
 2. 8. An oligonucleotide that hybridizes to a sequence recited in SEQ ID NO:1-149 under moderately stringent conditions.
 9. A method for stimulating and/or expanding T cells specific for a tumor protein, comprising contacting T cells with at least one component selected from the group consisting of: (a) polypeptides according to claim 2; (b) polynucleotides according to claim 1; and (c) antigen presenting cells that express a polypeptide according to claim 2, under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.
 10. An isolated T cell population, comprising T cells prepared according to the method of claim
 9. 11. A composition comprising a first component selected from the group consisting of physiologically acceptable carriers and immunostimulants, and a second component selected from the group consisting of: (a) polypeptides according to claim 2; (b) polynucleotides according to claim 1; (c) antibodies according to claim 5; (d) fusion proteins according to claim 7; (e) T cell populations according to claim 10; and (f) antigen presenting cells that express a polypeptide according to claim
 2. 12. A method for stimulating an immune response in a pancreatic cancer patient, comprising administering to the patient a composition of claim
 11. 13. A method for the treatment of a pancreatic cancer in a patient, comprising administering to the patient a composition of claim
 11. 14. A method for determining the presence of a pancreatic cancer in a patient, comprising the steps of: (a) obtaining a biological sample from the patient; (b) contacting the biological sample with an oligonucleotide according to claim 8; (c) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; and (d) compare the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence of the cancer in the patient.
 15. A diagnostic kit comprising at least one oligonucleotide according to claim
 8. 16. A diagnostic kit comprising at least one antibody according to claim 5 and a detection reagent, wherein the detection reagent comprises a reporter group.
 17. A method for inhibiting the development of a pancreatic cancer in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with at least one component selected from the group consisting of: (i) polypeptides according to claim 2; (ii) polynucleotides according to claim 1; and (iii) antigen presenting cells that express a polypeptide of claim 2, such that T cell proliferate; (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a pancreatic cancer in the patient. 